Free solution measurement of molecular interactions by backscattering interferometry

ABSTRACT

Disclosed are methods, systems, and apparatuses for the free solution measurement of molecular interactions by backscattering interferometry. In one aspect, the invention relates to method and systems for detecting molecular interaction between analytes in free-solution wherein the analytes are label-free and detection is performed by back-scattering interferometry. Also disclosed are label-free, free-solution, and/or real-time measurements of characteristic properties and/or chemical events using the disclosed techniques. The disclosed methods can have very low detection limits and/or very low sample volume requirements. Also disclosed are various biosensor applications of the disclosed techniques. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/857,953, filed Apr. 5, 2013, which is a continuation of U.S.application Ser. No. 12/674,610, filed Oct. 4, 2011, which is a nationalstage application of PCT/US2008/077145, filed Sep. 20, 2008, whichclaims the benefit of U.S. Application No. 60/973,829, filed Sep. 20,2007, which applications are hereby incorporated herein by reference inentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. R01EB003537-01A2 awarded by National Institutes of Health. The UnitedStates government has certain rights in the invention.

BACKGROUND

Capillary-based analysis schemes, biochemical analysis, basic researchin the biological sciences such as localized pH determinations intissues and studies in protein folding, detection and study ofmicroorganisms, and the miniaturization of instrumentation down to thesize of a chip all require small volume detection. With the advent oflasers, light sources possessing unique properties including highspatial coherence, monochromaticity and high photon flux, unparalleledsensitivity and selectivity in chemical analysis has become possible;these technologies, however, can be both expensive and difficult toimplement. In contrast, refractive index (RI) detection has beensuccessfully applied to several small volume analytical separationschemes. For various reasons, RI detection represents an attractivealternative to fluorescence and absorbance: it is relatively simple, itcan be used with a wide range of buffer systems, and it is universal,theoretically allowing detection of any solute, making it particularlyapplicable to solutes with poor absorption or fluorescence properties.

Conventional methods of probing intermolecular interactions typicallyrequire the use of one or more surface immobilized analytes in theinteraction as well as the use of chemical labels on one or bothanalytes. Surface immobilized methods are cumbersome due to theextraordinary effort required to optimize immobilization protocols aswell as their inherently high false positive and false negative bindingdetection rates, due to unwanted forces contributed by the supportingsubstrate. Moreover, these conventional methods typically fail toachieve detection at low detection limits or with low sample volumerequirements.

However, there remains a need in the art for systems and methods forfree-solution, label-free detection of intermolecular interactionsbetween analytes, preferably with low detection limits and/or low samplevolume requirements.

SUMMARY

As embodied and broadly described herein, the invention, in one aspect,relates to methods comprising detecting molecular interaction betweenanalytes in free-solution wherein the analytes are label-free anddetection is performed by back-scattering interferometry.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;introducing a second sample comprising a second non-immobilized analyteto be analyzed into the channel; allowing the first analyte to interactwith the second analyte to form one or more interaction products;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the fluid sample; and detecting positional shifts inthe light bands.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed, the channel containing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; and detecting positional shifts inthe light bands.

In various aspects, detecting positional shifts in the light bands cancomprise determining a change in a physical or chemical property of thefluid sample corresponding to the formation of the one or moreinteraction products of the first analyte with the second analyte. Forexample, the methods can further comprise the step of determining theformation of the one or more interaction products of the first analytewith the second analyte from the positional shifts of the light bands inthe interference patterns.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;introducing a second sample comprising a second non-immobilized analyteto be analyzed into the channel; allowing the first analyte to interactwith the second analyte to form one or more interaction products;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns.

In a further aspect, the invention relates to an interferometricdetection system comprising a substrate; a channel formed in thesubstrate for reception of a fluid sample to be analyzed; means forintroducing a first sample comprising a first analyte; means forintroducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel. In one aspect, the characteristic property is the formationof one or more interaction products of the interaction of the firstanalyte with the second analyte.

In a further aspect, the invention relates to a detection systemcomprising a microfluidic channel formed in a substrate; a solutioncomprising label-free analytes in free solution in the channel; and aninterferometer that detects molecular interactions between the analytesin the channel.

In a further aspect, the invention relates to a detection systemcomprising a solution comprising label-free analytes in free solution,wherein one of the analytes is present in a concentration of less thanabout 5.0×10⁻⁷M; and a detector that detects molecular interactionsbetween the analytes in the solution.

In a further aspect, the invention relates to the products of thedisclosed methods.

In a further aspect, the invention relates to chemical andbiotechnological (e.g., nucleic acid biosensors, enzyme biosensors,cellular biosensors, measurement of end-point values, determination ofkinetic parameters, immobilized bait measurements, free solutionmeasurements, label-free molecular interactions, bioassays, and thelike) applications employing the disclosed devices, systems, andmethods.

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a disclosedmethod or system does not specifically state that the steps are to belimited to a specific order, it is no way intended that an order beinferred, in any respect. This holds for any possible non-express basisfor interpretation, including matters of logic with respect toarrangement of steps or operational flow, plain meaning derived fromgrammatical organization or punctuation, or the number or type ofaspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 shows a schematic block diagram of an interferometric detectionsystem that is constructed in accordance with a first preferred aspectof the present invention.

FIG. 2 shows a diagrammatic illustration of a silica or other materialchip having a channel therein that forms a part of the system of FIG. 1,and is employed for receiving a sample whose refractive index orrefractive index related characteristic properties are to be determined.

FIGS. 3A and 3B show sectional views of the chip of FIG. 2 showing theshape of the channel, with FIG. 3A being taken along line 1-1 of FIG. 2,and FIG. 3B being taken along line 2-2 of FIG. 2.

FIG. 4 is an illustration of an interference fringe pattern that isproduced by the system of FIG. 1.

FIG. 5 is a schematic illustration of a second preferred aspect of thepresent invention that is employed for measuring the flow rate of a flowstream.

FIG. 6 is a schematic illustration of the interaction of an incidentlaser beam on the curved channel of the system of FIG. 1.

FIG. 7 is a schematic block diagram of another aspect of the inventionin which all of the system elements are formed on a single microchip.

FIG. 8 shows a schematic view of a suitable Back-ScatteringInterferometry (BSI) experimental setup seen in isometric view (A) andin plan view from the top (B) with a typical interference pattern shownat (C).

FIG. 9 is a view of the cross-section of the capillary of FIG. 8 showingthe ray paths through the capillary system.

FIG. 10 shows CCD camera images of a typical BSI interference pattern(A, B).

FIG. 11 shows line profiles (A, B) corresponding to interferencepatterns A, B respectively of FIG. 10.

FIG. 12 shows model predictions of the angle dependent variations in thereflected light intensity in the plane perpendicular to the capillarytube of FIG. 8.

FIG. 13 shows a calculated pattern for a smaller refractive index rangefrom a 100 μm ID/165 μm OD/12 μm coating capillary.

FIG. 14 shows experimentally obtained data from a 100 μm ID/165 μm OD/12μm coating capillary showing the low frequency variations of theinterference pattern as the refractive index is varied.

FIG. 15 shows experimentally obtained data for dilute sucrose solutionsin a 100 μm ID/165 μm OD/12 μm coating capillary.

FIG. 16 shows a calculated pattern for a 542 μm ID/673 μm OD/24 μmcoating capillary as function of reflection angle and refractive indexof the liquid.

FIG. 17 shows an experimentally obtained pattern for a 542 μm ID/673 μmOD/24 μm coating capillary.

FIG. 18 shows experimentally obtained data from a 542 μm ID/673 μm OD/24μm coating capillary.

FIG. 19 shows model low frequency component plots for three differentrefractive index samples.

FIG. 20 shows a bar graph showing the change in absolute signal formouse Actin DNA hybridization reactions using an OCIBD constructed inaccordance with the present invention: ssDNA corresponds to a singlestrand DNA immobilized on the surface and PBS buffer present in thechannel; cDNA corresponds to complete hybridization reaction when ssDNAand its complimentary cDNA are on the surface and PBS buffer is in thechannel.

FIG. 21 shows a bar graph showing the change in the signal produced byrepetitive hybridization and denaturation of mouse Actin DNA moleculesimmobilized on the surface. Variation in the signal between runs can beattributed to incomplete cDNA removal.

FIG. 22 shows A) Experimental setup for BSI, B) Microfluidic chip withserpentine mixer and restriction, C) Photograph of representative fringepatterns showing a RI induced position shift of the fringes, a cartoonrepresentation of a binding event and observed signal for a control andreactive pair binding event.

FIG. 23 shows A) Real-time association plots are shown for PA bindingIgG at various nanomolar concentrations. Extracted rates are plottedversus [IgG] in the inset. B) Steady-state values are plotted as afunction of IgG concentration and analyzed by Prism software.

FIG. 24 shows association curves of CaM with A) Ca²⁺, B) TFP, C)Calcineurin, and D) M13.

FIG. 25 shows IL-2—Ab binding curves with interaction assay performed incell free media.

FIG. 26 shows a) equations for binding of sHSP to the substrate: (1) T4Ltransition from native state (N) to unfolded protein (U), (2)dissociation of the sHSP large oligomer into dimers or tetramers, and(3) formation of the sHSP/T4L complex. Steady-state BSI data (♦) showsthat the magnitude of the binding signal increases with higherconcentrations of T4L-L99A. The linear rise in starting values (▪)reflects the response of BSI to increased concentrations of free L99A.c) The slope of the starting values of the traces in c) is identical tothat obtained from direct injection of T4L without αB-D3. (d) Thecalibration curve was used as a baseline subtraction to obtain acorrected steady-state binding trace. Isothermal titration calorimetry(ITC) analysis αB-D3⋅T4L-L99A binding. (e) Heat evolved after eachinjection (10 μL of T4L-L99A) was detected for 25 injections. f) Thearea under the curve was extracted and plotted against the molar ratioto obtain a binding isotherm. Non-linear least-squares analysis was usedto determine thermodynamic parameters.

FIG. 27 shows a) the structure of T4L highlighting the sites ofmutation. b) BSI signals following the injection of T4L mutants withoutsHSP. Increasing T4L concentration shifts the baseline but does not leadto a time-dependent change, ruling out mixing artifacts. c) Raw BSI datashow an overlap in the fringe patterns of the mutants demonstrating BSIis insensitive to differences in their stabilities. A zoomed in regionof the interference patterns is shown and compared to a fringe patternfrom a buffer solution demonstrating the sensitivity of the instrumentto changes in refractive index.

FIG. 28 shows graphs of kinetics of αB-D3 binding to T4L-D70N (a) andT4L-L99A-A130S (b) monitored by BSI (black) with kinetic traces fit viaglobal analysis (red). Analysis of the steady-state data (c) shows thatthe magnitude of binding as detected by BSI for αB-D3⋅T4L-L99A-A130S issignificantly greater than seen with αB-D3⋅T4L-D70N. As a control, αB-D3was assayed against multiple concentrations of WT-T4L, exhibiting nobinding across the concentration range. [T4L-D70N]=1, 2.5, 5, 10, 20,30, 40, 60, and 90 μM. [T4L-L99A-A130S]=1, 2.5, 5, 10, 15, 20, 40, 60,80, and 100 μM.

FIG. 29 shows (a) Interaction of αA-R49C-crystallin with multipleconcentrations of βB1-crystallin at physiologically relevant conditionsas detected by BSI. Corrected steady-state values (b) matched well withthose obtained from a global analysis of the kinetic data (c).

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which mayneed to be independently confirmed.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a substrate,” “apolymer,” or “a sample” includes mixtures of two or more suchsubstrates, polymers, or samples, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where the event or circumstanceoccurs and instances where it does not.

As used herein, the term “polymer” refers to a relatively high molecularweight organic compound, natural or synthetic (e.g., polyethylene,rubber, cellulose), whose structure can be represented by a repeatedsmall unit, the monomer (e.g., ethane, isoprene, (3-glucose). Syntheticpolymers are typically formed by addition or condensation polymerizationof monomers.

As used herein, the term “copolymer” refers to a polymer formed from twoor more different repeating units (monomer residues). By way of exampleand without limitation, a copolymer can be an alternating copolymer, arandom copolymer, a block copolymer, or a graft copolymer.

As used herein, the term “bioassay” refers to a procedure fordetermining the concentration, purity, and/or biological activity of asubstance.

As used herein, the term “chemical event” refers to a change in aphysical or chemical property of an analyte in a sample that can bedetected by the disclosed systems and methods. For example, a change inrefractive index (RI), solute concentration and/or temperature can be achemical event. As a further example, a biochemical binding orassociation (e.g., DNA hybridization) between two chemical or biologicalspecies can be a chemical event. That is, a chemical event can be theformation of one or more interaction products of the interaction of afirst analyte with a second analyte. As a further example, adisassociation of a complex or molecule can also be detected as an RIchange. As a further example, a change in temperature, concentration,and association/dissociation can be observed as a function of time. As afurther example, bioassays can be performed and can be used to observe achemical event.

As used herein, the terms “equilibrium constant” and “Kc” and “Keq”refer to the ratio of concentrations when equilibrium is reached in areversible reaction. For example, for a general reaction given by theequation:

aA+bB

cC+dD,

the equilibrium constant can be expressed by:

$K_{c} = {\frac{{\lbrack C\rbrack^{c}\lbrack D\rbrack}^{d}}{{\lbrack A\rbrack^{a}\lbrack B\rbrack}^{b}}.}$

An equilibrium constant can be temperature- and pressure-dependent buthas the same value, irrespective of the amounts of A, B, C, and D. Aspecific type of equilibrium constant that measures the propensity of alarger object to separate (dissociate) reversibly into smallercomponents is a “dissociation constant” or “Kd.” A dissociation constantis the inverse of an “affinity constant.”

As used herein, the terms “dissociation rate” is a concentrationdependent quantity and involves the “dissociation rate constant” or“K_(D).” The dissociation rate constant relates the rate at whichmolecules dissociate to the concentration of the molecules. Adissociation can be described as AB→A+B, and the rate of dissociation(dissociation rate) is equal to K_(D)[AB]. In general, the larger thevalue of K_(D), the faster the inherent rate of dissociation.

As used herein, the terms “association rate” is a concentrationdependent quantity and involves the “association rate constant” and“K_(A).”

The association rate constant relates the rate at which moleculesassociate to the concentration of the molecules. An association can bedescribed as A+B→AB, and the rate of association (association rate) isequal to K_(A)[A][B]. In general, the larger the value of K_(A), thefaster the inherent rate of association.

As used herein, the term “free-solution” refers to a lack of surfaceimmobilization.

As used herein, the term “label-free” describes a detection methodwherein the detectability of an analyte is not dependent upon thepresence or absence of a detectable label. For example, “label-free” canrefer to the lack of a detectable label. It is understood that theability of a label to be detected can be dependent upon the detectionmethod. That is, an analyte having a moiety capable of serving as adetectable label for a first detection method can be considered“label-free” when a second detection method (wherein the label is notdetectable) is employed.

As used herein, the term “detectable label” refers to any moiety thatcan be selectively detected in a screening assay. Examples includewithout limitation, radiolabels, (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I)affinity tags (e.g. biotin/avidin or streptavidin), binding sites forantibodies, metal binding domains, epitope tags, FLASH bindingdomains—See U.S. Pat. Nos. 6,451,569; 6,054,271; 6,008,378 and5,932,474—glutathione or maltose binding domains) fluorescent orluminescent moieties (e.g. fluorescein and derivatives, GFP, rhodamineand derivatives, lanthanides etc.), and enzymatic moieties (e.g.horseradish peroxidase, β-galactosidase, β-lactamase, luciferase,alkaline phosphatase). Such detectable labels can be formed in situ, forexample, through use of an unlabeled primary antibody which can bedetected by a secondary antibody having an attached detectable label.Further examples include imaging agents such as radioconjugate,cytotoxin, cytokine, Gadolinium-DTPA, a quantum dot, iron oxide, andmanganese oxide.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc., of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specific aspector combination of aspects of the methods of the invention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. Interferometric Detection

In one aspect, the invention relates to an interferometric detectionsystem and method that can be used, for example, for detection ofrefractive index changes in picoliter sized volumes for chip-scaleanalyses. The detection system has numerous applications, including theobservation and quantification of molecular interactions, molecularconcentrations, bioassays, universal/RI detection for CE (capillaryelectrophoresis), CEC (capillary electrochromatography) and FIA (flowinjection analysis), physiometry, cell sorting/detection by scatter,ultra micro calorimetry, flow rate sensing, and temperature sensing.

Thus, in one aspect, the invention fulfills a need for a new sensingmethodology applicable to μ-TAS (micro Total Analysis Systems) throughprovision of an interferometric detection system and method thatcircumvent the drawbacks of conventional interferometric methods and thelimitations of the forward scatter technique. The system includes asource of coherent light, such as a diode or He—Ne laser, a channel ofcapillary dimensions that is preferably etched or molded in a substratefor reception of a sample to be analyzed, and a photodetector fordetecting backscattered light from the sample at a detection zone.

In one aspect, the laser source generates an easy to align simpleoptical train comprised of collimated laser beam that is incident on theetched channel (or capillary) for generating the backscattered light.The backscattered light comprises interference fringe patterns thatresult from the reflective and refractive interaction of the incidentlaser beam with the channel walls and the sample. These fringe patternsinclude a plurality of light bands whose positions shift as therefractive index of the sample is varied, either through compositionalchanges or through temperature changes, for example. The photodetectordetects the backscattered light and converts it into intensity signalsthat vary as the positions of the light bands in the fringe patternsshift, and can thus be employed to determine the refractive index (RI),or an RI related characteristic property, of the sample. A signalanalyzer, such as a computer or an electrical circuit, is employed forthis purpose to analyze the photodetector signals, and determine thecharacteristic property of the sample.

In one aspect, the channel has a generally semi-circular cross-sectionalshape. A unique multi-pass optical configuration is inherently createdby the channel characteristics, and is based on the interaction of theunfocused laser beam and the curved surface of the channel, that allowsinterferometric measurements in small volumes at high sensitivity.Additionally, if a laser diode is employed as the source, not only doesthis enable use of wavelength modulation for significant improvements insignal-to-noise ratio, but it also enables integration of the entiredetector device directly onto a single microchip.

Alternatively, the channel can have a substantially circular orgenerally rectangular cross-sectional shape. In one aspect, thesubstrate and channel together comprise a capillary tube. In a furtheraspect, the substrate and channel together comprise a microfluidicdevice, for example, a silica substrate, or a polymeric substrate [e.g.,polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA)], and anetched channel formed in the substrate for reception of a fluid sample,the channel having a cross sectional shape. It is also contemplated thatthe substrate can further comprise a reference channel. For example, thedetection system can employ a second channel, which can comprise acapillary or an on-chip channel of semi-circular or rectangularcross-section.

The detector can be employed for any application that requiresinterferometric measurements; however, the detector is particularlyattractive for making universal solute quantification, temperature andflow rate measurements. In these applications, the detector providesultra-high sensitivity due to the multi-pass optical configuration ofthe channel. In the temperature measuring aspect, the signal analyzerreceives the signals generated by the photodetector and analyzes themusing the principle that the refractive index of the sample variesproportionally to its temperature. In this manner, the signal analyzercan calculate temperature changes in the sample from positional shiftsin the detected interference fringe patterns.

In the flow measuring aspect, the same principle is also employed by thesignal analyzer to identify a point in time at which perturbation isdetected in a flow stream in the channel. In the case of a thermalperturbation, a flow stream whose flow rate is to be determined, islocally heated at a point that is known distance along the channel fromthe detection zone. The signal analyzer for this aspect includes atiming means or circuit that notes the time at which the flow streamheating occurs. Then, the signal analyzer determines from the positionalshifts of the light bands in the interference fringe patterns, the timeat which thermal perturbation in the flow stream arrives at thedetection zone. The signal analyzer can then determine the flow ratefrom the time interval and distance values. Other perturbations to theflow stream, include, but are not limited to, introduction into thestream of small physical objects, such as glass microbeads ornanoparticles. Heating of gold particles in response to a chemicalreaction or by the change in absorption of light due to surface-boundsolutes or the capture of targets contained within the solution can beused to enhance the temperature induced RI perturbation and thus tointerrogate the composition of the sample.

In one aspect, the fluid sample is a liquid, which can be asubstantially pure liquid, a solution, or a mixture (e.g., biologicalfluids, cellular fluids). In a further aspect, the fluid can furthercomprise one or more analytes.

1. Free-Solution Determination Methods for Molecular Interactions

In contrast to conventional techniques that observe immobilizedanalytes—which necessarily limit conformational and translationalfreedom for analytes and are, thus, in vitro measurements—free-solutionanalysis techniques mimic in vivo measurements, because analytes enjoyunrestricted freedom in all three dimensions during measurement.

In one aspect, the invention relates to a method comprising detectingmolecular interaction between analytes in free-solution wherein theanalytes are label-free and detection is performed by back-scatteringinterferometry. In one aspect, the analytes are present in a sample in amicrofluidic channel in a substrate. In a further aspect,back-scattering interferometry comprises directing a coherent light beamonto the substrate such that the light beam is incident on the channelto generate backscattered light through reflective and refractiveinteraction of the light beam with a substrate channel interface and thesample, the backscattered light comprising interference fringe patternsincluding a plurality of spaced light bands whose positions shift inresponse to changes in the refractive index of the fluid sample.

In a further aspect, the invention relates to a method comprisingdetecting molecular interaction between analytes in free-solution,wherein the analytes are label-free, wherein one of the analytes ispresent in a concentration of less than about 5.0×10⁻⁷M. In one aspect,the analytes are present in a sample in a microfluidic channel in asubstrate. In a further aspect, detection is performed byback-scattering interferometry.

It is contemplated that the method can be used to determine one or moreof an equilibrium constant, a dissociation constant, a dissociationrate, a dissociation rate constant, an association rate, and/or anassociation rate constant of the interaction.

Each of the one or more analytes can be introduced into the channel in asample. Two or more analytes can be present in the same or in differentsamples. Each of the one or more analytes can independently be presentin a suitable concentration, for example, a concentration of less thanabout 5.0×10⁻⁷M, of less than about 1.0×10⁻⁷M, a concentration of lessthan about 5.0×10⁻⁸M, of less than about 1.0×10⁻⁸M, of less than about5.0×10⁻⁹M, of less than about 1.0×10⁻⁹M, of less than about 1.0×10⁻¹⁰M,of less than about 5.0×10⁻¹⁰M, of less than about 5.0×10⁻¹¹M, of lessthan about 1.0×10⁻¹¹M, of less than about 5.0×10⁻¹²M, or of less thanabout 1.0×10⁻¹²M.

In one aspect, the interaction can be a biomolecular interaction. Forexample, two analytes can associate to provide an interaction product(e.g., adduct, complex, or new compound). In a further aspect, ananalyte can dissociate to provide two or more interaction products. Infurther aspects, more than two analytes can be involved in theinteraction.

In one aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;introducing a second sample comprising a second non-immobilized analyteto be analyzed into the channel; allowing the first analyte to interactwith the second analyte to form one or more interaction products;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns.

The disclosed techniques can determine the interaction between one ormore analytes by monitoring, measuring, and/or detecting the formationand/or steady state relative abundance of one or more analyteinteraction products from the interaction of the one or more analytes.The determination can be performed qualitatively or quantitatively.Interaction rate information can be derived from various measurements ofthe interaction.

In a further aspect, the first sample is combined with the second sampleprior to introduction. That is, the analytes are combined (andpotentially interacting) prior to performing the disclosed methods. Inthis aspect, the step of introducing the first analyte and the step ofintroducing the second analyte are performed simultaneously.

In a further aspect, the first sample is combined with the second sampleafter introduction. That is, the analytes can be combined at a pointbefore the channel, or at a point within the channel, when performingthe disclosed methods. In this aspect, the step of introducing the firstanalyte and the step of introducing the second analyte are performedeither simultaneously or sequentially. In a further aspect, thedetecting step is performed during the interaction of the first analytewith the second analyte.

In an alternative mode of operation, the method can involve end-pointmeasurement. That is, the method can determine the occurrence and/orcompleteness of an interaction between two or more analytes that havebeen mixed prior to analysis. Thus, in a further aspect, the inventionrelates to a method for free-solution determination of molecularinteractions comprising the steps of providing a substrate having achannel formed therein for reception of a fluid sample to be analyzed;introducing a first sample comprising a first non-immobilized analyte tobe analyzed into the channel; establishing a baseline interferometricresponse by directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the first sample; introducing a second samplecomprising a mixture of the first non-immobilized analyte and a secondnon-immobilized analyte to be analyzed, wherein the first analyte tointeracts with the second analyte to form one or more interactionproducts, into the channel; directing a coherent light beam onto thesubstrate such that the light beam is incident on the channel togenerate backscattered light through reflective and refractiveinteraction of the light beam with a substrate/channel interface and thesample, the backscattered light comprising interference fringe patternsincluding a plurality of spaced light bands whose positions shift inresponse to changes in the refractive index of the second sample;detecting positional shifts in the light bands relative to the baseline;and determining the formation of the one or more interaction products ofthe first analyte with the second analyte from the positional shifts ofthe light bands in the interference patterns.

A first sample (e.g., a solution including a first non-immobilizedanalyte to be analyzed) can be introduced into the channel of thesubstrate. The first sample can be provided having a known concentrationof the first analyte.

A baseline interferometric response can then be established by directinga coherent light beam onto the substrate such that the light beam isincident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the first sample.

A second sample (e.g., a solution including a mixture of the firstnon-immobilized analyte and a second non-immobilized analyte to beanalyzed, wherein the first analyte to interacts with the second analyteto form one or more interaction products) can then be introduced intothe channel. In various aspects, the second sample can be provided as apre-mixed sample of the first non-immobilized analyte and the secondnon-immobilized analyte or provided by adding a sample comprising thesecond non-immobilized analyte to the first sample. In one aspect, thefirst sample is a solution of the first analyte, which is displaced inthe channel by the introduction of the second sample, which is asolution of both the first analyte and the second analyte. The secondsample can be provided having a known concentration of the firstanalyte, which can be the same as the concentration of the first analytein the first solution. The second sample can also be provided having aknown concentration of the second analyte.

A coherent light beam can then directed onto the substrate such that thelight beam is incident on the channel to generate backscattered lightthrough reflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample.

Positional shifts in the light bands relative to the baseline can thenbe detected, and the interaction of the first analyte with the secondanalyte can then be determined from the positional shifts of the lightbands in the interference patterns. The rate of interaction between thetwo analytes can thus be monitored, thereby determining the quantity ofone of the binding partners, having a priori knowledge of the abundanceof its other binding partner. That is, the system and method provides asignal (i.e, positional shifts in the light bands) that is proportionalto abundance of the analyte.

In a further aspect, the first analyte and/or the second analyte is/areunlabeled. While the disclosed methods can be used in connection withunlabeled analytes, it is contemplated that the analytes can beoptionally labeled. Such labeling can be convenient for preceding,subsequent, or simultaneous analysis by other analytical methods.

In a further aspect, the interaction is the formation of one or morecovalent bonds, electrostatic bonds, hydrogen bonds, or hydrophobicinteractions. In a further aspect, the interaction creates aconformational change in at least one of the analytes. In a furtheraspect, the interaction is a binding event between one or more ofantibody-antigen, protein-protein, small molecule-small molecule; smallmolecule-protein, drug-receptor; antibody-cell; protein-cell;oligonucleotide-cell; carbohydrate-cell; cell-cell; enzyme-substrate;protein-DNA; protein-aptamer; DNA-DNA; RNA-RNA; DNA-RNA; protein-RNA;small molecule-nucleic acid; biomolecule-molecular imprint;biomolecule-protein mimetic; biomolecule-antibody derivatives;lectin-carbohydrate; biomolecule-carbohydrate; small molecule-micelle;small molecule-cell membrane; and enzyme-substrate.

In a further aspect, a fluid sample can comprise at least one of aliquid or a gas. In particular aspects, a fluid sample comprises asolution of one or more analytes and one or more liquid solvents. Asolution can be provided in an organic solvent or in water. In certainaspects, the solution can comprise man-made preparations or naturallyoccurring substances. In certain aspects, the solution can comprise abody fluid (e.g., peripheral blood, urine, cerebrospinal fluid,pulmonary lavage, gastric lavage, bile, vaginal secretions, seminalfluid, aqueous humor, and vitreous humor) from a human, a mammal,another animal, or a plant.

Generally, the substrate and channel can comprise any material suitablefor containing and providing a sample for analysis and capable of beinginterrogated by the coherent light beam to generate backscattered lightthrough reflective and refractive interaction of the light beam with asubstrate/channel interface and the sample. In one aspect, the substrateand channel together comprise a capillary tube. In a further aspect,wherein the substrate and channel together comprise a microfluidicdevice.

In a further aspect, the microfluidic device comprises a polymericsubstrate and an etched channel formed in the substrate for reception ofa fluid sample, the channel having a cross sectional shape. In a furtheraspect, the polymeric substrate can be selected from rigid andtransparent plastics. In various further aspects, the polymericsubstrate comprises one or more polymers selected from polycarbonate,polydimethylsiloxane, fluorosilicone, polytetrafluoroethylene,poly(methyl methacrylate), polyhexamethyldisilazane, polypropylene,starch-based polymers, epoxy, and acrylics.

In a further aspect, the microfluidic device comprises a silicasubstrate and an etched channel formed in the substrate for reception ofa fluid sample, the channel having a cross sectional shape, which can besubstantially circular, substantially semi-circular, or substantiallyrectangular, as disclosed herein.

It is contemplated the substrate can comprise one or more than onechannel. In a further aspect, the substrate further comprises areference channel.

The disclosed methods can provide real-time, free-solution detection ofmolecular interactions with very low detection limits. That is, in oneaspect, the invention relates to a method for real-time, free-solutiondetermination of molecular interactions comprising the step of detectingthe formation of one or more interaction products of two unlabeled,non-immobilized analytes, wherein at least one of the analytes ispresent during the determination at a concentration of less than about5.0×10⁻⁵M. In various further embodiments, the concentration can be lessthan about 1.0×10⁻⁵M, for example, less than about 5.0×10⁻⁶M, less thanabout 1.0×10⁻⁶M, less than about 5.0×10⁻⁷M, less than about 1.0×10⁻⁷M,less than about 5.0×10⁻⁸M, less than about 1.0×10⁻⁸M, less than about5.0×10⁻⁹M, or less than about 1.0×10⁻⁹M. In a further aspect, theconcentration can be less than about 5.0×10⁻¹⁰M, for example, less thanabout 1.0×10⁻¹⁰M, less than about 5.0×10⁻¹¹M, less than about1.0×10⁻¹¹M, less than about 5.0×10⁻¹²M, or less than about 1.0×10⁻¹²M.

The disclosed methods can provide real-time, free-solution detection ofmolecular interactions with very low sample volume requirements. Thatis, in one aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL. In various furtherembodiments, the sample volume can be less than about 250 nL, forexample, less than about 100 nL, less than about 10 nL, less than about1 nL, less than about 500 pL, less than about 250 pL, or less than about100 pL.

In an even further aspect, the disclosed methods further comprise thestep of performing a chromatographic separation and/or anelectrophoretic separation on the sample before, during, or after thedetermining the characteristic property step.

2. Interferometric Detection Systems for Free-Solution Determination ofMolecular Interactions

In one aspect, the invention relates to an interferometric detectionsystem comprising a substrate; a channel formed in the substrate forreception of a fluid sample to be analyzed; means for introducing afirst sample comprising a first analyte; means for introducing a secondsample comprising a second analyte; optionally, means for mixing thefirst sample and the second sample; a coherent light source forgenerating a coherent light beam, the light source being positioned todirect the light beam onto the substrate such that the light beam isincident on the channel to thereby generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the fluid sample; a photodetector for receiving thebackscattered light and generating one or more intensity signals thatvary as a function of positional shifts of the light bands; and a signalanalyzer for receiving the intensity signals, and determining therefrom,a characteristic property of the fluid sample in the channel.

In one aspect, the invention relates to a detection system comprising amicrofluidic channel formed in a substrate; a solution comprisinglabel-free analytes in free solution in the channel; and aninterferometer that detects molecular interactions between the analytesin the channel. In a further aspect, the invention relates to adetection system comprising a solution comprising label-free analytes infree solution, wherein one of the analytes is present in a concentrationof less than about 5.0×10⁻⁷M; and a detector that detects molecularinteractions between the analytes in the solution. The detector can be,for example, an interferometer. Likewise, detection can be, for example,by means of back-scattering interferometry. The channel can be formedby, for example, etching, by molding, by micromachining, or byphotolithography. It is contemplated that the system can be used todetermine one or more of an equilibrium constant, a dissociationconstant, a dissociation rate, a dissociation rate constant, anassociation rate, and/or an association rate constant of an interaction.

In various aspects, a means for introducing a sample can be anyapparatus, system, or construct capable of conveying a sample into thesystem and/or directing a sample into or through the channel such thatthe light beam incident on the channel encounters at least a portion ofthe sample, thereby generating backscattered light through reflectiveand refractive interaction of the light beam with a substrate/channelinterface and the sample. Examples of means for introducing a sampleinclude an opening in a capillary tube, an injection port, a secondcapillary tube in fluid communication with the substrate and channel, amicrofluidic channel in fluid communication with the substrate andchannel, a syringe, a pipette, a chromatographic separation apparatus influid communication with the substrate and channel, and/or anelectrophoretic separation apparatus in fluid communication with thesubstrate and channel.

It is understood that the means for introducing a first sample and themeans for introducing a second sample can comprise the same or adifferent means. It is also understood that the means for introducing afirst sample and the means for introducing a second sample can comprisethe same type (e.g., both are injection ports) or a different type(e.g., one is a capillary tube and the other is a syringe) of means.

In various aspects, a means for mixing can be any means for apparatus,system, or construct capable of combining two samples such that thesamples are in intimate contact and capable of interacting physicallyand/or chemically. Examples of means for mixing include a blender, asonication apparatus, a microfluidic serpentine mixer, and amicrofluidic restriction.

In a further aspect, the coherent light source is a laser, for example aHe/Ne laser, a VCSEL laser, or a diode laser.

In a further aspect, the system can further comprise a referencechannel. In a further aspect, the substrate and channel togethercomprise a capillary tube. In a further aspect, the substrate andchannel together comprise a microfluidic device. In a further aspect,the microfluidic device comprises a silica substrate and an etchedchannel formed in the substrate for reception of a fluid sample, thechannel having a cross sectional shape. In a further aspect, themicrofluidic device comprises a glass substrate and an etched channelformed in the substrate for reception of a fluid sample, the channelhaving a cross sectional shape. In a further aspect, the cross sectionalshape is substantially rectangular, substantially circular, or generallysemi-circular. In a still further aspect, the microfluidic devicecomprises a polymeric substrate and an etched channel formed in thesubstrate for reception of a fluid sample, the channel having a crosssectional shape. In a further aspect, the polymeric substrate can beselected from rigid and transparent plastics. In various furtheraspects, the polymeric substrate comprises one or more polymers selectedfrom polycarbonate, polydimethylsiloxane, fluorosilicone,polytetrafluoroethylene, poly(methyl methacrylate),polyhexamethyldisilazane, polypropylene, starch-based polymers, epoxy,and acrylics.

Thus, in various aspects, the channel is formed by etching, by molding,by micromachining, or by photolithography.

With reference now to one aspect of the invention, an interferometricdetection system 10 is illustrated in FIG. 1 which makes use of atechnique that employs backscattered light to determine the RI or RIrelated characteristic properties of a sample. The backscatter detectiontechnique is generally disclosed in U.S. Pat. No. 5,325,170 to Bornhop,which is hereby incorporated by reference. More recently, the techniqueis referred to as Back-Scatter Interferometry or BSI.

The interferometric detection system 10 includes a laser or other sourceof coherent light 12, which is preferably a low power (3-15 mW) laser(He/Ne or Diode), and generates a laser beam 14. As with anyinterferometric technique for micro-chemical analysis, BSI benefits frommany of advantages lasers provide, including high spatial coherence,monochromaticity, and high photon flux. The intensity of the laser beam14 can be reduced as needed with a series of optional neutral densityfilters 16 (e.g., optical density of 0.5, 1.0, 0.3 respectively). Uponreduction of the intensity, the beam 14 is directed to an optionalmirror 18 that is angled at approximately 45° with respect to the planeof propagation of the laser beam 14. The mirror 18 re-directs the beam14 onto a substrate chip 20 having a channel 22 formed therein,preferably by etching, for reception of a sample volume to be analyzed.It will be understood that the mirror 18 can be deleted, and the laser12 can be repositioned to aim the laser beam 14 directly at the etchedchannel 22 if desired.

The chip 20 is preferably formed of silica or glass, but can be anyother suitable optically transmissive material, such as plastic (i.e.,polymeric material). One requirement, however, is that the material fromwhich the chip 20 is made, must have a different index of refractionthan that of a sample volume to be tested. In the exemplary aspect ofFIG. 1, the chip 20 is shown mounted on a peltier temperature controlledA1 support block 23, which in turn is affixed to an X-Y translationstage 24 that allows adjustment of the chip 20 relative to the laserbeam 14. More particularly, the chip 20 is preferably tilted slightly(e.g., approximately 7°) so that the (nearly direct) backscattered lightfrom the channel 22 can be directed onto a photodetector 25. The purposeof the temperature controlled support block 23 is to insure that thesample in the channel 22 is maintained at a constant temperature sincethe RI of a sample is known to vary linearly with its temperature.Alternatively, this characteristic also allows the detection system 10to be utilized for making very accurate temperature measurements.

The photodetector 25 can be one of any number of image sensing devices,including a bi-cell position sensor, a linear or area array CCD or CMOScamera and laser beam analyzer assembly, a slit-photodetector assembly,an avalanche photodiode, or any other suitable photodetection device.The backscattered light comprises interference fringe patterns thatresult from the reflective and refractive interaction of the incidentlaser beam 14 with the walls of the channel 22 and the sample. Thesefringe patterns include a plurality of light bands (see FIG. 4) whosepositions shift as the refractive index of the sample is varied, eitherthrough compositional changes or through temperature changes, forexample. The photodetector 25 detects the backscattered light andconverts it into one or more intensity signals that vary as thepositions of the light bands in the fringe patterns shift. For fringeprofiling, the photodetector 25 is preferably mounted above the chip 20at an approximately 45° angle thereto.

The intensity signals from the photodetector 25 are fed into a signalanalyzer 28 for fringe pattern analysis, and determination therefrom ofthe RI or an RI related characteristic property of a sample in thechannel 22. The signal analyzer 28 can be a computer (e.g., a PC) or adedicated electrical circuit, for example. Preferably, the signalanalyzer 28 includes the programming or circuitry necessary to determinefrom the intensity signals, the RI or other characteristic properties ofthe sample to be determined, such as temperature or flow rate, forexample.

FIG. 2 shows a top view of the chip 20 showing the channel 22. Aninjection port 30 and an exit port 32 are laser drilled at opposite endsof the channel 22 to allow for introduction and removal of a fluidsample to be analyzed. The laser beam 14 is directed to impinge upon thechannel 22 at a point 34 that is a short distance (e.g., about 2 mm)from the exit port 32 and is graphically shown by the circle, labeled“detection zone,” in FIG. 2.

As illustrated in FIGS. 3A and 3B, the chip 20 preferably consists offirst and second substrate pieces 36 and 38 that are fused together,with the channel 22 being formed in the first, top substrate piece 36,and having a generally semicircular cross-sectional shape. Thesemicircle has a radius R of between 5 and 150 microns, and mostpreferably between 10 and 50 microns. Although it is preferred that thechannel 22 be truly semicircular in shape, to accommodate conventionaletching techniques, the channel 22 is formed by first etching a first 90degree arc 40 in the top substrate piece 36, and then etching a second90 degree arc 42. This etching process inherently results in theformation of a short, flat portion or segment 44 between the first andsecond arcs 40 and 42. The length L of the flat portion 44 should be asshort as possible, preferably 5-25 microns. When the length of the flatportion 44 does not exceed the length of the channel radius R, there isno adverse effect on the interference fringes that are generated by thechannel 22. The second, bottom substrate piece 38 forms a floor 45 ofthe channel 22, and has a thickness T that is approximately one third toone times the radius R of the arcs 40 and 42.

Even though the channel 22 can be of the general shape of a semi-circle(half circle), including the flat portion 44, relatively high contrastinterference fringes (much like those seen with full capillaries) havebeen observed in experiments on a prototype of the invention. Theinherent characteristics of the channel 22 result in a multi passoptical configuration in which multipath reflections occur, and increasethe sensitivity of the detector system 10. A typical interferencepattern produced by an unmodified chip filled with distilled/deionizedwater is shown in the false color intensity profile (black no photonsand white is the intensity for detector saturation) shown in FIG. 4.These observations can be important because, 1) the features (arcs 40and 42) on the chip 20 that produce the interference fringes are quitecommon and easy to manufacture, 2) no additional optics are needed, and3) the fringes have very high contrast allowing sensitive detection ofoptical path length changes. It is noteworthy that all of themeasurements are obtained using a very simple optical train with noadditional focusing or collection optics and using a chip that has noreflective coatings. In short, the chip-scale RI detector configurationcan use unaltered chips.

Numerous experiments have been conducted to verify the operation of theon-chip detection system 10, and determine which components provide thebest sensitivity. A first experiment using varying concentrations of aglycerol solution was performed to evaluate the detection system 10using the CCD camera for recording and measuring fringe movement. Thefringe movement varies linearly with concentration (change in RI) over 2decades. The limit of detection calculated at 3δ was 31.47 mM of soluteand was limited mainly by the LBA (Laser Beam Analyzer from Spiricon,Inc. Logan, Utah) software used at that time to read the camera.

In a further experiment, to improve the sensitivity of the on-chip RImeasurement, the neutral density filters 16 were removed and aslit/photodetector assembly was used instead of the CCD camera/laserbeam analyzer system. In this experiment, the slit/photodetectorassembly was located on the order of 28 cm from the front surface of thechip 20. The photodetector 25 consisted of a pin photodiode integratedwith a 632.8 nm interference filter (Coherent-Ealing) wired with asimple current to voltage circuit. A 50-micron precision air slit(Melles Griot) was mounted vertically in the center of the activesurface area of the photodiode. The voltage output from the photodiodewas then amplified (Gain=100) by a low-noise preamplifier (StanfordResearch Systems) using a 30 Hz low pass filter (12 dB/octave). Theanalog signal from the preamplifier was then digitized with an externalDAQ board (PPIO-AIO8, CyberResearch, Branford, Conn.) and displayed onthe PC computer 28 running a digital strip-chart recorder (Labtech forWindows).

The slit-photodetector assembly was aligned on the edge of a fringe inorder to monitor fringe movement. The position of the assemblycorresponds to the edge of the sloping intensity gradient of the workingfringe and is located at I=1/e² of the intensity distribution. Since theintensity of a backscattered fringe is essentially Gaussian, a change inrefractive index of the solution in the probe volume produces a changein the light intensity striking the active surface of the photodetector25. As the fringe shifts, a small voltage output from the photodetector25 is observed, which is linearly proportional to a change in refractiveindex (Δn).

A calibration curve was generated with the slit/photodetector using theexact same procedure and glycerol solutions of the same concentrationsas with the CCD/LBA configuration. The 3δ detection limit for thebackscatter detector using a slit/photodetector assembly was found to be18.33 mM, substantially better than the 31.47 mM limit achieved with theCCD/LBA experimental set up. The lower detection limits are achievablewith a slit/photodetector assembly since small positional shifts of thebackscattered fringes result in large intensity changes due to theirpseudo-Gaussian intensity profile. The CCD/laser beam analyzer systemmeasures only positional shifts, which are considerably less sensitivethan the intensity changes seen by the slit/photodetector assembly.

In a further experiment, to improve the signal-to-noise ratio of themeasurement still further, the photodetector 25 was a small areaavalanche photodiode (e.g., such as those available from TexasOptoelectronics, Inc.). The avalanche photodiode (APD) was operated nearthe breakdown voltage and driven with a reverse bias. The APD wasaligned on the edge of the fringe as described for theslit/photodetector assembly, and fringe movement was denoted by changesin intensity. The signal from the APD was digitized with an external DAQboard (PPIO-AIO8, CyberResearch, Branford, Conn.) and displayed on thePC computer 28 running a digital stripchart recorder (Labtech forWindows).

Running tests on a series of glycerol solutions revealed that the 3δdetection limit for glycerol is just 4.1 mM. By using the APD (even at awavelength, 632.8 nm, where the device has poor quantum efficiency) a4.4 fold S/N gain is realized.

Still further increases in sensitivity have been realized in subsequentexperiments using a bi-cell position photodetector, and a diode laserwith special optics to produce a pseudo-Gaussian beam of approximately75 μm, at a distance of 50 cm and over a relatively long focal length.In this study the detection volume was 188 picoliters and a 2δconcentration detection limit for glycerol of 494 μM (139×10⁻¹⁵ moles or12.8 picograms of solute) was attained, without active thermal control.Thus, a reduction in the volume and an increase in sensitivity wererealized as a consequence of several technical modifications to thesystem.

The detection limits achieved in the foregoing experiments represent thelowest RI detection limits that have been achieved to date with a systemthat is compatible with chip-scale sensing (low nanoliter detectionvolumes). For reference, BSI is already an order of magnitude moresensitive than the holographic forward scatter technique.

It is noteworthy that these detection limits were accomplished withoutany active thermal control of the chip (resulting in increased noise dueto thermal perturbations in the dc mode (i.e., no wavelengthmodulation)) and using minimal active electronic filtering. Inmeasurements of refractive index (n), the primary source of noise isthermal sensitivity. For most cases involving fluids, n has a relativelyhigh thermal coefficient (dn/dT), requiring very precise temperaturestabilization of the system. As an example, dn/dT for H₂O is on theorder of 8×10⁻⁴° C.⁻¹, so at an analytically useful detection limit forΔn of one part in 10⁶, the temperature-induced signal corresponds to achange in T of 1×10⁻²° C. Therefore, thermal stability of the systemmust be maintained at the millidegree centigrade label, to determine nto one part in 10⁸. This level of temperature control can be achievedusing a thermostated flow cell with active control using a Peltierthermoelectric cooling chip (e.g., such as is available from Melcore,Trenton, N.J.) controlled by a power supply (e.g., ILX Lightwave,Bozeman, Mont.) wired in feedback from a calibrated thermocouple.

Conversely, as discussed previously, thermal “noise” in RI measurementscan be used to the advantage of the analyst. For example, thermalsensitivity can be used to determine minute temperature changes insmall-volume following streams, non-invasive process stream monitoring,and even protein folding. The relationship between do and dT is linear.Therefore, BSI can be used to measure thermal changes at a microdegreecentigrade level and to determine dn/dT for fluids.

To demonstrate use of the system 10 of FIG. 1 for detecting temperaturechanges, another experiment was conducted. In this experiment,thermometry was performed in a probe volume of just 3.14×10⁻⁹ L asdefined by the diameter of the laser beam 14 and the radius (in thiscase, 50 microns) of the etched channel 22. Distilled/deionized waterwas hydrodynamically injected into the channel 22 and allowed totemperature- and pressure-stabilize. Next the temperature of the channel22 was manually changed in approximately 0.3° C. increments, the samplewas allowed to temperature stabilize, and a relative change inrefractive index measurement was obtained. Upon graphing the results ofrelative change in RI versus temperature for water, a detection limit of0.011° C. (11 millidegree C.) was determined based on the 3 sigmastatistics. These results confirm that the signal analyzer 28 can beprogrammed to determine the temperature of the sample from an analysisof the fringe pattern signals with a high degree of sensitivity.

A further aspect of the present invention is illustrated in FIG. 5. Thisaspect is designed for measuring the flow rate of a flow stream flowingthrough the channel 22. The signal analyzer 28 in this aspect containstiming circuitry or programming, and controls operation of a heatingsource 50 that provides localized heating of a point 52 along thechannel 22 that is spaced a known distance x from the detection zone 34.Preferably, the heating source is an infrared laser that can providerapid localized heating of a sample flow stream in the channel 22.

In the operation of this aspect, the heating source 50 is triggered at afirst instant in time to provide the localized heating of a portion ofthe flow stream. This creates a temperature perturbation in the flowstream that moves toward the detection zone 34. The signal analyzer 28then monitors the intensity signals generated by the photodetector 25,and detects therefrom, the instant in time when the temperatureperturbation arrives at the detection zone 34. The time interval betweenwhen the flow stream was heated and when the temperature perturbation isdetected is then employed with the value of x to determine the flow rateof the flow stream.

Using ASAP (an optical modeling program from Breault Research, TucsonAriz.) a few preliminary modeling experiments were performed todemonstrate the multi pass optical configuration provided by the channel22, and the path length insensitivity that results. In the firstinvestigation illumination impinges onto the etched side of the chip 20,so that the light impinges on the curved surface just after entering thesubstrate. FIG. 6 illustrates the results of this simulation, andclearly shows the multipath reflections that increase the system'ssensitivity, or leads to an inherent insensitivity of performance on thesize of the channel 22. Put another way, the multi pass configurationeliminates optical path length constraints, thus allowing for smallerand smaller detection volumes. In FIG. 6, nine initial rays are tracedthrough a chip with an etched channel with a diameter of 100 μm. Thelaser source is located at some distance in +Z direction. Splits (thenumber of rays that will continue at interfaces) are set to 3. Themiddle plane simulates the lid that covers the channel. Since the indexrefraction on both sides of that plane is the same its presence does notaffect the rays intersecting that plane. Since the rays that continue totravel in the −Z direction, after they passed through the chip, do notcontribute to the formation of the backscattered fringe pattern, theycan be ignored and dropped out of simulation.

Even lower detection limits for BSI can be achieved. First, simplyincreasing the distance of the photodetector 25 from the front surfaceof the etched channel 22 can produce larger “apparent” fringe movementbecause angular displacement grows as the detector to channel distanceincreases. In general, this geometric relationship dictates sensitivityto angular displacement and indicates that every two-fold increase indistance can produce at least a two-fold sensitivity improvement.Second, lower detection limits can be achieved by using either a longerwavelength laser or an APD whose sensitivity is maximized at thewavelength of the laser used. For example, at the He/Ne wavelength of632.8 nm, the radiant responsivity of the current detector isapproximately 10 A/W, but at the wavelength of 700 nm, the radiantresponsivity of the device increases by a factor of three to 30 A/W. Asa result, detection limits are predicted to improve by at leastthree-fold. Third, the detection volume for BSI on a chip can be furtherreduced by using a smaller diameter laser beam (e.g., lasers generating10 μm diameter beams are available), or a fiber couple diode lasercombined with a smaller radius channel.

With respect to the detector system 10, while HeNe lasers have excellentoptical properties, they can be limited in applications that demandminiaturization by their bulky size. As a result, VCSELs and diodelasers are replacing HeNe lasers in many industrial, medical, andanalytical applications. VCSELs and diode lasers, in general, are solidstate, low-cost compact, light sources that possess many of theproperties of gas lasers (HeNe's). Among them are good beam quality(TEM₀₀), low divergence, and some polarization purity. Furthermore, theyhave characteristic long lifetimes (in excess of 50,000 hours), andprovide reasonable coherent lengths (as great as 1 meter). VCSELs anddiode lasers can differ, however, from HeNe lasers in several ways,particularly when using them as interferometry sources. First,wavelength stability of most VCSELs and diode lasers is generally poordue to the device's structure (small cavity size), resulting in adependency on and sensitivity to current and temperature changes.Second, while emitting light that is inherently linearly polarized, thepolarity purity of a VCSEL's or diode laser's beam is relatively low(100:1). Nevertheless, if proper care is taken, VCSELs and diode lasersare low cost, coherent light sources that are adequate forinterferometric detection schemes such as BSI for both RI andpolarimetric detection.

One advantage of VCSELs and diode lasers is that they can facilitatereduction in size of the RI detector system 10 to the point of beingincorporated directly onto the chip 20. FIG. 7 illustrates such anaspect in which both the laser 12 and the photodetector 25 are formedintegrally with the chip 20.

Another advantage of using VCSELs or laser diodes in interferometry isthat their optical output (wavelength) can be easily modulated throughthe supply current. Wavelength modulation opens a path to potentialalternative detection schemes in on-chip RI detection usingmicro-interferometry as a method of decreasing thermal sensitivity ofthe measurement and lowering the limit of detection of the technique.Thus, the system 10 can be configured so that detection is performed inthe AC regime (source wavelength modulation).

When wavelength modulation techniques are used with VCSELs and diodelasers, it is possible to make exceeding sensitive optical absorbancemeasurements. In fact the sensitivity possible approaches the shot noiselimit, i.e. 10⁻⁷ AU in a 1 Hz bandwidth. Furthermore, with the advent ofrapidly tunable (over a wide wavelength range), single mode, circularbeam VCSELS, these devices are suitable sources for the on-chipinterferometric detection technique. In short, by using such an approachfor on-chip RI detection based on micro-interferometry, a significant(as much as 500-fold) improvement in S/N can be achievable for theinstrument.

Using on-chip RI detection based on micro-interferometry, the disclosedinvention performs interference detection in channels with ultra-smallvolumes and with a simple optical configuration that requires noadditional optics. The on-chip RI detector is an effective universaldetection system that expands the ability to sense or quantitativelydetect otherwise invisible solutes, particularly those important toclinical diagnostics, proteomic, genomic and metabolomic analysis andhigh throughput molecular drug screening. The detector's S/N ratio isnot hindered by volume reduction, its probe volume and detection volumeare the same, it is a non-invasive method, and is universal in nature.Thus, the detector can play an important role in integrated-omicstechnology, drug discovery and development and diagnostic medicine. Itcan also allow protein folding and biochemical bonding measurementspreviously not possible. Reaction kinetics can be followed in nanolitervolumes, and millidegree temperature changes can be quantified. Finally,the invention allows the further development of μ-TAS and othertechniques for cellular level analysis and bioassays and clinicaldiagnostic testing.

C. Back-Scattering Interferometry

Rapid monitoring and quantitative detection of ultra small volumesamples is in great demand. One analytical approach, Back-ScatteringInterferometry (BSI), derives from the observation that coherent lightimpinging on a cylindrically shaped capillary produces a highlymodulated interference pattern. Typically, BSI analyses reflections froma capillary tube filled with a liquid of which one wants to measure therefractive index. First used and described by Bornhop et al. [Bornhop,D. J. Appl. Opt., 1995, 34, 3234 3239; Bornhop, D. J. U.S. Pat. No.5,325,170, 1994; Swinney, K; Markov, D; Bornhop, D. J., Review ofScientific instruments, 2000, 71, 2684 2692.], the technique has beenshown capable of measuring changes in refractive index of liquids on theorder of 10⁻⁷. The BSI technique is a simple and universal method ofdetecting refractive index changes in small volumes of liquid and can beapplied to monitor changes in concentrations of solutes, flow rates andtemperature, all conducted in nanoliter volumes.

The BSI technique is based on interference of laser light after it isreflected from different regions in a capillary or like samplecontainer. Suitable methods and apparatus are described in U.S. Pat. No.5,325,170 and WO-A-01/14858, which are hereby incorporated by reference.The reflected or back scattered light is viewed across a range of angleswith respect to the laser light path. The reflections generate aninterference pattern that moves in relation to such angles upon changingrefractive index of the sample. The small angle interference patterntraditionally considered has a repetition frequency in the refractiveindex space that limits the ability to measure refractive index torefractive index changes causing one such repetition. Such refractiveindex changes are typically on the order of three decades.

Accordingly, the disclosed invention provides a method for performing ameasurement of refractive index comprising directing a coherent lightbeam along a light path to impinge on a first light transmissivematerial and pass there through, to pass through a sample which is to bethe subject of the measurement, and to impinge on a further lighttransmissive material, the sample being located between the first andfurther materials, detecting reflected light over a range of angles withrespect to the light path, the reflected light including reflectionsfrom interfaces between different substances including interfacesbetween the first material and the sample and between the sample and thefurther material which interfere to produce an interference patterncomprising alternating lighter and darker fringes spatially separatedaccording to their angular position with respect to the light path, andconducting an analysis of the interference pattern to determine therefrom the refractive index, wherein the analysis comprises observation ofa parameter of the interference pattern which is quantitatively relatedto sample refractive index dependent variations in the intensity ofreflections of light which has passed through the sample.

In accordance with preferred variants of this procedure, the analysiscomprises one or both of: (a) the observation of the angle with respectto the light path at which there is an abrupt change in the intensity ofthe lighter fringes, or (b) the observation of the position of thesefringes of a low frequency component of the variation of intensitybetween the lighter and darker fringes. The first of these (a), reliesupon the dependency of the angle at which total internal reflectionoccurs at an interface between the sample and the further material onthe refractive index of the sample. The second (b), relies upon thedependency of the intensity of reflections from that interface on therefractive index as given by the Fresnel coefficients. The rectangularchips also have a single competent from diffraction at the corners.

The first material and the further material are usually composed of thesame substance and may be opposite side walls of a container withinwhich the sample is held or conducted. For instance, the sample may becontained in, e.g. flowed through, a capillary dimensioned flow channelsuch as a capillary tube. The side wall of the capillary tube nearer thelight source is then the “first material” and the opposite side wall isthe “further material.” The cross-sectional depth of the channel islimited only by the coherence length of the light and its breadth islimited only by the width of the light beam. Preferably, the depth ofthe channel is from 1 to 10 um, but it may be from 1 to 20 um or up to50 um or more, e.g. up to 1 mm or more. However, sizes of up to 5 mm or10 mm or more are possible. Suitably, the breadth of the channel is from0.5 to 2 times its depth, e.g., equal to its depth.

In one aspect, at least one the interface involving the sample at whichlight is reflected is curved in a plane containing the light path, thecurved interface being convex in the direction facing the incoming lightif it is the interface between the first material and the sample andbeing concave in the direction facing the incoming light if it is theinterface between the sample and the further material. Preferably, thesample is in a channel of substantially circular, generallysemi-circular, or rectangular cross-section.

The sample is typically a liquid, and can be flowing or stationary.However, the sample can also be a solid or a gas in various aspects ofthe present invention. The first and/or further materials will normallybe solid but in principle can be liquid, e.g., can be formed by asheathing flow of guidance liquid(s) in a microfluidic device, with thesample being sheathed flow of liquid between such guidance flows. Thesample may also be contained in a flow channel of appropriate dimensionsin substrate such as a microfluidic chip. The method may therefore beemployed to obtain a read out of the result of a reaction conducted on a“lab on a chip” type of device.

In contrast to conventional, the invention can, in one aspect, make useof observations of the interference pattern at large angles with respectto the light path, e.g., the range of angles can include angles up to atleast 10°, at least 15°, at least 20°, at least 25°, or at least 30° orcan include angles of at least 35°, at least 40°, at least 45°, at least50°, at least 55°, or at least 60°.

The invention includes apparatus for use in performing a method asdescribed, which apparatus comprises a source of spatially coherentlight, a sample holder for receiving a sample upon which to perform themethod positioned in a light path from the light source, a detector fordetecting light reflected from the sample over a range of angles withrespect to the light path, and data processing means for receivingmeasurements of light intensity from the detector and for conducting ananalysis thereon, wherein the analysis comprised determining a parameterof an interference pattern produced by the reflected light whichparameter is quantitatively related to sample refractive index dependentvariations in the intensity of reflections of light which has passedthrough the sample.

The data processing means can be adapted to perform an analysis whichcomprises one or both of: (a) the determination of the angle withrespect to the light path at which there is an abrupt change in theintensity of the lighter fringes, or (b) the determination of theposition of fringes of a low frequency component of the variation ofintensity between the lighter and darker fringes. In a further aspect,the data processing means further comprises a processor programmed todetermine a characteristic property of the fluid sample in the channelby performing a method comprising the steps of: computing an overlappingproduct of signal A and signal B generated from the detector, andassigning values to elements of a list R based on the overlappingproduct; summing a set of elements from R to produce a value q;multiplying a set of elements from R by an odd function; summing one ormore products from the multiplying step to produce a value p; andcalculating the shift between signal A and signal B as a function of pdivided by q.

The apparatus can comprise means for controlling the temperature of thesample, e.g., a heater and/or a Peltier cooler and a temperaturemeasuring device. As would be readily understood by one of skill, theterm “back-scatter” is generally used to describe the origin of thelight rays that form the interference pattern. On the basis oftheoretical analysis of the origin of the interference pattern presentedherein, the term “reflection” is more strictly accurate, but thephenomenon referred to by these terms is in each case the same.

In one aspect, the source of coherent light is a laser, suitably a He—Nelaser or a diode laser or VCSEL. The laser light may be coupled to thesite of measurement by known wave-guiding techniques or may beconventionally directed to the measurement site by free spacetransmission.

The measured refractive index can be indicative of a number ofproperties of the sample including the presence or concentration of asolute substance, e.g. a reaction product, pressure, temperature or flowrate (e.g., by determining when a thermal perturbation in a liquid flowreaches a detector).

In one aspect, the detector is a CCD array of suitable resolution.

The invention includes apparatus as described herein, wherein the sampleholder is configured to allow a sample to flow there through and whereinthe sample holder is connected to receive a separated sample from asample separation device in which components of a mixed sample areseparated, e.g., by capillary electrophoresis, capillaryelectrochromatography or HPLC. Accordingly, viewed from anotherperspective, the invention provides chromatography apparatus having arefractive index measuring unit as described herein as a detector.

More generally, the sample holder of the apparatus described above canbe a flow through passage so that the contents of the channel may becontinuously monitored to observe changes in the content thereof. Thesechanges may include the temporary presence of cells and the out flowfrom the sample holder may be diverted to a selected one of two or moreoutlet channels according to the measurements of RI observed in thesample holder, e.g., to achieve sorting of cells in response to suchmeasurements. The sample holder can contain a stationary analyticalreagent (e.g., a coating of an antibody, oligonucleotide or otherselective binding agent) and changes in the refractive index caused bythe binding of a binding partner to the reagent may be observed. In viewof the small sample size which it is possible to observe, the sampleholder can contain a biological cell and metabolic changes therein maybe observed as changes in the refractive index of the cell.

In one aspect, the invention relates to a method for performing ameasurement of refractive index comprising directing a coherent lightbeam along a light path to impinge on a first light transmissivematerial and pass there through, to pass through a sample which is to bethe subject of the measurement, and to impinge on a further lighttransmissive material, the sample being located between the first andfurther materials, detecting reflected light over a range of angles withrespect to the light path, the reflected light including reflectionsfrom interfaces between different substances including interfacesbetween the first material and the sample and between the sample and thefurther material which interfere to produce an interference patterncomprising alternating lighter and darker fringes spatially separatedaccording to their angular position with respect to the light path, andconducting an analysis of the interference pattern to determine therefrom the refractive index, wherein the analysis comprises observation ofa parameter of the interference pattern which is quantitatively relatedto sample refractive index dependent variations in the intensity ofreflections of light which has passed through the sample. The firstmaterial and the further material can comprise the same substance ordifferent substances. The sample can be contained in a flow channelhaving a cross-sectional depth of, for example, up to 1 mm in thedirection of the light path. For example, the sample can be contained ina capillary tube.

In a yet further aspect, the analysis comprises one or both of: (a) theobservation of the angle with respect to the light path at which thereis an abrupt change in the intensity of the lighter fringes, or (b) theobservation of the position of these fringes of a low frequencycomponent of the variation of intensity between the lighter and darkerfringes. In a still further aspect, at least one the interface involvingthe sample at which light is reflected is curved in a plane containingthe light path, the curved interface being convex in the directionfacing the incoming light if it is the interface between the firstmaterial and the sample and being concave in the direction facing theincoming light if it is the interface between the sample and the furthermaterial.

In one aspect, the invention relates to an apparatus for use inperforming a measurement of refractive index, which apparatus comprisesa source of coherent light, a sample holder for receiving a sample uponwhich to perform the method positioned in a light path from the lightsource, the sample holder providing a first interface between the sampleholder and a sample receiving space in the sample holder and a secondinterface between the sample receiving space and the sample holder, thefirst and second interfaces being spaced along the light path, adetector for detecting light reflected in use from a the sample over arange of angles with respect to the light path, the reflected lightincluding reflections from the first and second interfaces whichinterfere to produce and interference pattern comprising alternatinglighter and darker fringes spatially separated according to theirangular position with respect to the light path, and data processingmeans for receiving measurements of light intensity from the detectorand for conducting an analysis thereon, wherein the analysis comprisesdetermining a parameter of the interference pattern produced by thereflected light which parameter is quantitatively related to samplerefractive index dependent variations in the intensity of reflections oflight which has passed through the sample.

In a further aspect, the data processing means is adapted to perform ananalysis which comprises: the determination of the position of thefringes of a low frequency component of the variation of intensitybetween the lighter and darker fringes.

In a further aspect, the data processing means is adapted to perform ananalysis which comprises the determination of the angle with respect tothe light path at which there is an abrupt change in the intensity ofthe lighter fringes.

In a further aspect, the data processing means is adapted to perform ananalysis which comprises the determination of the angle with respect tothe light path at which there is an abrupt change in the intensity ofthe lighter fringes and comprises the determination of the position ofthe fringes of a low frequency component of the variation of intensitybetween the lighter and darker fringes.

In a further aspect, the data processing means is adapted to perform ananalysis which comprises one or both of: (a) the determination of theangle with respect to the light path at which there is an abrupt changein the intensity of the lighter fringes, or (b) the determination of theposition of these fringes of a low frequency component of the variationof intensity between the lighter and darker fringes, and wherein thesample holder locates the sample between a first material defining thefirst interface with the sample and a further material defining thesecond interface with the sample, which first and second materials arecomposed of the same substance.

In a further aspect, the sample holder is so constructed that at leastone of the first and the second interfaces is curved in a planecontaining the light path, the curved interface being convex in thedirection facing the incoming light if it is the interface nearer thelight source in the light path and being concave in the direction facingthe incoming light if it is the interface more distant from the lightsource in the light path.

A typical BSI scheme as previously known is shown in FIG. 8. The systemconsists of a laser 110 that impinges its beam on a capillary tube 112filled with a liquid of which one wants to measure refractive index,thereby creating an interference pattern 114. This interference pattern,which changes with changes in the liquid's refractive index, is thenmeasured using a CCD detector 116. A typically observed interferencepattern in the reflection direction is seen in C. This is analyzed bydata processing means 120.

The demonstrated sensitivity of 10⁻⁷ is reached by following thedisplacement of the individual light fringes of the interference patternwithin 0.3 angular degrees [Swinney, K; Markov, D; Bornhop, D. J; Reviewof Scientific instruments, 2000, 71, 2684 2692.] from the directly backreflected direction, as one changes the refractive index of the liquid.The fringe pattern is periodic in refractive index space with a periodof the order of 10⁻³. This limits the dynamic measurement range to theorder of 10⁻³, which for many purposes requires additional knowledgeabout the absolute value of the refractive index.

As the measurement monitors a displacement of the fringe pattern, it isinherently a differential measurement, employing calibration both forthe absolute level of the refractive index as well as for thedifferential factor. This factor describes the fringe movementcorresponding to a given change in the refractive index.

The dynamic range of the BSI system may be increased by taking intoaccount other variations of the interference pattern with changingrefractive index than those previously considered. The dynamic range isincreased without compromising the high differential sensitivitypreviously reported [Swinney, K; Markov, D; Bornhop, D. J; Review ofScientific instruments, 2000, 71, 2684 2692.]. Theoretical descriptionof the BSI scheme has been improved to include an extended optical raytracing model that matches the range in angular and refractive indexspace of the experiments, thus providing new information about thestructure of the reflected light interference pattern. In contrast tothe previously proposed model [Tarigan, H. J; Neill, P. Kenmore, C. K;Bornhop, D. J. Anal. Chem., 1996, 68, 1762 1770.], this model is capableof explaining all frequency components that appear in the interferencepattern. Furthermore, the model has been used to predict an abruptchange in the intensity of the reflected light interference fringes,which depends uniquely on the absolute value of the refractive index ofthe probed sample. Moreover, this feature has been experimentallyconfirmed. The improved understanding of the BSI system provides twopreferred approaches to an absolute measurement of the refractive indexof samples, which are preferably liquids in the refractive index rangebetween water (1.33) and glass (1.50). The first approach is based onthe measurement of the depth of modulation of the interference patterncaused by variations in the Fresnel coefficients. The second approach isbased on the measurement of the total internal reflection angle withinthe capillary or other sample container.

Previously systems of similar geometry to the BSI scheme have beenmodeled by obtaining solutions to Maxwell's equations governing lightpropagation [Pedrotti, F. L; Pedrotti, L. S. Introduction to optics, 2nded.; Prentice-Hall New Jersey, 1996; Chapter 27.] or by optical raytracing. Kerker and Matijevic [Kerker, M; Matijevic, E. J. Opt. Soc.Am., 1961, 51, 506-508.] made the first complete model based onsolutions to Maxwell's equations describing two concentric cylinders.Watkins confirmed these results experimentally [Watkins, L. S; J. Opt.Soc. Am., 1974, 64, 767-772.]. However Watkins considered optical glassfibers with thick claddings and therefore obtained results significantlydifferent to those observed in BSI, as the interference pattern isdescribed not to be dependent on the refractive index of the core in theback-scatter angle regime. Marcuse and Presby [Marcuse. D; Presby, H. M.J. Opt. Soc. Am., 1975, 65, 367 375.] extended this model to also takeinto account the case of a thin cladding of the fibers. From theirresults, an abrupt change in the back-scattered light intensity patternis observed. However, it was not realized that this abrupt change couldbe utilized to obtain the absolute refractive index with high precision,since they were attempting to determine the outer radius of the glassfiber, and they were not concerned with the core index. The position ofthe abrupt change depends on the core index. Horton and Williamson[Horton. R; Williamson, W. J. J. Opt. Soc. Am., 1973, 63, 1204 1210.]made a ray tracing model of an optical fiber obtaining information aboutthe ratio between inner and outer radii of the fiber. The approach theyused is a back calculation assuming a planar wave front of the output.The rays considered in their model are not the same as in the presentmodel, as they consider fibers with a thick cladding, and make use ofmultiple reflections inside the cladding. This is due to the fact thatthey use significantly different refractive indices of the core thanthose considered in BSI. The BSI system has been modeled using aray-tracing model by H. Tarigan et al. [Tarigan, H. J; Neill, P.Kenmore, C. K; Bornhop, D. J. Anal. Chem., 1996, 68, 1762-1770.].However, their model is limited by considering only small angleback-scattered light (0.3 degrees).

The present model has been extended to include reflection angles up to90 (side reflections). This is done in the geometric optics regime byusing Snell's law:

n _(i)×sin(θ_(i))=n _(j)×sin(θ_(j))  (1)

where n_(i) and n_(j) are the refractive indices of the media and θ_(i)and θ_(j) are the angles of light propagation in the respective media.Furthermore the law of reflection, |θ_(in)|=|θ_(out)|, is used. Forangles beyond a few degrees it is not possible to use the assumption(sin θ≈θ) done by Tarigan et al. This implies that a simple analyticalequation cannot be obtained. The present model traces six beams, seeFIG. 9, through the system and calculates their interference in adetection plane placed in the far field region. For each type of beam anumber of rays (typically 1000) are traced. The information carriedalong with each ray is its position, angle, intensity and phase. At thedetection plane the interference is calculated based on the informationpackages of all rays. The six beams considered in the model interfereby:

I _(ij)=2√(I _(i) ×I _(j))×cos(ρ_(i)−ρ_(j))  (2)

where I is the intensity and ρ is the phase of each individual ray, andi and j are indices for each ray, respectively. The model is developedto also take into account the polymer coating on the capillary, thusrequiring six beams. The model assumes circular geometry of thecapillary and that the laser can be described by plane waves.

The model assumption of circular geometry of the capillary is justifiedby the observation that no significant change in the pattern wasobserved during rotation of the capillary (TSP100170, PolymicroTechnologies) along the capillary axis. The tilt of the wave front fromthe laser (05-LHR-HeNe, Melles Griot) was analyzed using a beam analyzer(CLAS-2D, Wave Front Sciences) and was observed to be less than 0.01micro radians, thus justifying the assumption of a planar wave front.The smallest spacing of refractive index changes is the thickness of thecoating of 12 micrometer.

Therefore the assumption of geometrical optics being adequate isjustified since the wavelength used (632.8 nm) is much smaller than thedistances otherwise present in the system.

In what follows, modeling and experimental work is based on the use ofapparatus as shown in FIG. 8. The BSI experiments were done by mountingthe capillary on a translation stage and making a He—Ne laser beamimpinge perpendicularly on the capillary. The reflected or backscatteredlight was collected using a screen and a CCD camera (C4742-95,Hamamatsu). Requirements for the laser include a coherence length of atleast about twice the diameter of the capillary and a wavelength atwhich the capillary is substantially transparent. Requirements for thedetector include high one-dimensional spatial resolution and an adequateintensity resolution, depending on the application. Passive temperaturecontrol consisting of a large thermal reservoir (an aluminum block)thermally connected to the capillary was used to stabilize temperature.Temperature fluctuations affect the refractive index of the liquidsubstantially. Active temperature control is only needed if thedetection of changes in refractive index of less than 10⁻⁵ is required.In this work, passive temperature stabilization is adequate, as therefractive index fluctuations in the system caused by temperaturefluctuations in a controlled environment are on the 10⁻⁵ scale,corresponding to 0.1° C. temperature fluctuations for water.

The capillaries used in the experiments were purchased from PolymicroTechnologies (AZ, US). Two sizes of capillaries have been used. Thedimensions of the capillaries are 100 μm inner diameter (ID), 165 μmouter diameter (OD) with a 12 μm thick polyimid coating (TSP100170) and542 μm ID, 673 μm OD with a 24 μm thick polyimid coating (TSP530660),respectively. In the experiments, the refractive index was changed from1.33 to 1.5 by using both sucrose (Sigma Chemicals Company) and NaSCN(Merck) aqueous solutions. The RI of the solutions was measured in arefractometer (RL3, Polskie Zaklady Optyczne, Warsaw) immediately afterthe sample had been injected into the capillary. A typical interferencepattern thus obtained is shown in FIG. 10A, with corresponding lineprofile of the intensity shown in FIG. 11A. In FIG. 10B two of thefringes in 3A are enlarged, showing a finer structure. A line profile ofthe intensity from 3B is seen in 4B. The visual appearance is enhancedby low pass filtering, a Fourier filter, of the raw data. The raw dataand the low pass filtered data have been offset for clarity. It is seenthat the intensity pattern contains multiple frequency components. Theperiod of the fringe pattern corresponding to medium frequency (MF)components is shown in FIG. 11B. Similarly, the period of the fringepattern corresponding to low frequency (LF) components is shown in FIG.11A. In the following, frequency components are referred to asfrequencies. Under certain circumstances, one is able to observe a moreclosely spaced component, or ripple, of the intensity profile, heredenoted high frequency (HF) variations. As one increases the refractiveindex of the liquid in the capillary, the intensity profile shiftstowards lower reflection angles, see FIG. 14. However, the highfrequency variation component is spatially fixed and does not move asone changes the refractive index, in accordance with previousobservations [Markov, D; Swinney, K; Norville, K; Lu, D; Bornhop, D. J.Electrophoresis, 2002, 23, 809 812.].

By investigating the experimentally observed frequencies of the fringepattern, it may be shown by geometrical considerations that the distancebetween the origin points of the interfering rays (points a through finFIG. 9) on the capillary are approximately five to ten times larger inthe high frequency case than in the medium frequency case. By usinggeometrical considerations, it is possible to calculate the distancebetween origins of the interfering rays for all frequency components. Itwas found that the distance required to produce the high frequencyvariations is on the order of the capillary diameter. This indicatesthat the rays responsible for this high frequency variation arescattered from the edges of the capillary, thereby not being affected bythe liquid within the capillary. This fits the observed behavior well,since the high frequency component is not observed to be displaced asone changes the refractive index of the liquid in the capillary.

The low frequency component is spatially stationary as well, since thiscomponent is caused by the common interference between the three raysreflected from the front of the capillary (points a through c in FIG.9), as well as the common interference between the three rays reflectedfrom the back of the capillary (points d through fin FIG. 9). Thedisplacement of this component is zero for the part originating from thefront, since these rays do not traverse the liquid and thus experiencethe same optical path length by different refractive indices of theliquid. For the second part the displacement is small, because all threerays experience almost the same change in optical path length traversedrelative to each other.

In contrast to the high and low frequency components, respectively, themedium frequency component originates from the interference between raysreflected at the front (points a c in FIG. 9), and at the back of thecapillary (points d through fin FIG. 9). These rays experience a largerelative change in optical path length traversed, as the rays from thefront do not experience a change in optical path length whereas the raysreflected from the back do. It is this relative change in the opticalpath length between different paths that causes the movement of themedium frequency component of the interference pattern as refractiveindex changes, yielding the ultra-high sensitivity previously described.

The results from the model are plotted in FIG. 12 and FIG. 13 asfunction of reflection angle and refractive index of the liquid for a100 μm ID/165 μm OD/12 um coating capillary. The results have beencompiled in this plot by stacking such line plots for closely spacedliquid refractive indices into a two-dimensional overview of thereflection behavior. This plot corresponds to 1643 injections of liquidwith different refractive indices. Bands of light (fringes) move towardslarger reflection angles as the refractive index is increased.Overlaying vertical band structures of higher and lower light intensityare seen. These structures do not move as the refractive index ischanged. An abrupt change in the intensity level (a) is seen movingtowards lower back-scattering angles for refractive indices of theliquid above 1.45. The grayscale represents the intensity of the patternin the given reflection angle for the given refractive index of theliquid in arbitrary units. In FIG. 13 the movement of the fringes at areflection angle of 20° is measured to be approximately 2.1° per 0.01refractive index change, measuring from (a) to (b).

Experimentally obtained data are plotted the same way as the model andthe results are shown in FIGS. 7 and 8. Here 25 measurements of theinterference pattern have been made, each at different refractive index.At each refractive index level a line profile of the interferencepattern has been obtained. These measurements have been stackedvertically into one figure. In FIG. 15, the refractive index intervalbetween measurements is less than the change required to move a fringeone fringe-width, thus allowing one to monitor the medium frequencyfringes as continuous bands. The movement of these fringes varies withreflection angle. For a reflection angle of 20 this movement is measuredto be 4.0 per 0.01 refractive index change, measuring from (a) to (b).Low frequency variations are seen as vertical light bands.

These fringes do not appear to form continuous bands in the verticalrefractive index dependent direction due to the large change inrefractive index between measurements. An abrupt change in the intensity(a) is seen for high refractive indices (above 1.43), which movestowards lower reflection angles for increasing refractive index.

At each refractive index a line profile of the intensity of theinterference pattern has been made. Each line profile has been extendedvertically. The extended line profiles have been stacked into a singleplot. These figures are used to directly compare the model and theexperiment. In the BSI experiments two sets of fringes are alwayspresent. The fringes moving outwards (medium frequency) with increasingrefractive index are measured to move 4.0° when the refractive index ischanged by 0.01 at a reflection angle of 20° (a, bin FIG. 15). The modelpredicts a movement of 2.1° (a, bin FIG. 13). These are the fringestraditionally used for measuring refractive index using the BSItechnique. This model predicts both low frequency and medium frequencyvariations of the pattern. These frequencies will be discussed below.The low frequency fringes are not moving significantly with changingrefractive index. The model predicts no movement of these fringes. Thenumber of these fringes in the model is 13 and in the experiment 10fringes are observed within a range of reflection angles from 14 to 54°.Both model and experiment shows an abrupt change in intensity at largereflection angles. This abrupt change in intensity is somewhat displacedin the modeled results compared to the experimental results, but it iswithin experimental error. The movement of this abrupt change inintensity in experiments qualitatively agrees with modeling of the BSIsystem—the model predicts the behavior of the BSI system qualitatively.The predictions of the model have been used to select the propercapillary dimensions for applications of the BSI technique.

A first preferred aspect of the invention performs absolute measurementof refractive index based on Fresnel coefficients. Even though the lowfrequency variations remain stationary in terms of reflection detectionangles, their intensity changes as the refractive index of the liquidchanges. As the intensity of the rays are in part determined by theFresnel coefficients of the surface of reflection, the system can beconfigured in such a way that the intensity of the low frequencycomponent can be used as a measure for the refractive index on a coarserscale. This may be done by either index matching the coating and theglass tubing, thereby eliminating the reflection from the coating-glasssurface (points b and e in FIG. 9) or by stripping the coating off thecapillary. The low frequency component is then caused by interferencebetween two rays; the ray reflected by the air-coating (points a and finFIG. 9) or air-glass interface (points b and e if the coating isremoved) and the ray reflected from the glass-liquid interface (points cand d in FIG. 9). Since the intensity of this last ray is determined bythe Fresnel coefficients of this surface consisting of glass withconstant refractive index and the liquid to be probed, the absolutevalue of the refractive index of the liquid may be calculated from therelative intensity of the two rays, which is given by the depth ofmodulation of the low frequency component. This is possible if therefractive index of the air, glass, and coating is known. If one wantsto measure depth of modulation to a certain degree, one needs at leastthis degree of intensity resolution in the detection system. Since theCCD camera used in these examples has 255 intensity levels, morerefractive index resolution than the difference in refractive indexbetween air and glass divided by the number of detectable intensitylevels, which corresponds to 5×10⁻³, cannot be acquired without thedisclosed improved methods. A camera with a larger number of intensitylevels can alternately be used.

However, FIG. 19 shows model LF plots (similar to that marked in FIG.10) calculated for liquids of three different refractive indices. It canbe seen that the lateral position of the fringes are dependent on therefractive index, but that the amplitude does not change based onrefractive index.

A second preferred aspect relies on the dependence on refractive indexof the critical angle at which total internal reflection occurs. Themodel predicts an abrupt change in intensity moving towards lowerreflection angles as the refractive index of the liquid approaches theone of the glass tubing, see line marked by (a) in FIG. 12. This featureof the interference pattern is also observed experimentally (see (a) inFIG. 14) and agrees with the predicted feature in position-refractiveindex space within experimental error. A feature similar to this hasbeen reported for optical glass fibers [Horton R.; Williamson, W. J.; J.Opt. Soc. Am., 1973, 63, 1204 1210.]. However these fibers have adifferent optical configuration, and the mechanisms responsible aredifferent. In the case of optical glass fibers the mechanism responsibleis grazing of a certain ray on the core of the fiber being dependent ofthe inner radius of the glass fiber. Without wishing to be bound bytheory, it is believed that the mechanism giving rise to the phenomenonin BSI is total internal reflection in the wall of the capillary, beingdependent on the refractive index of the liquid in the capillary. Themain source of error is the dimensions of the capillary, which have anuncertainty of 6 μm for the 100 μm ID/165 μm OD/12 um coating capillaryaccording to the manufacturer. The way of determining the absolute valueof refractive index on a coarser scale is to look at this feature of thepattern. Both the model and the experiment show an abrupt change inlight intensity at higher reflection angles, and the position of thischange varies with refractive index. However, using a 100 μm ID/165 pmOD capillary this change takes place at refractive indices 1.40 to 1.50,which is not the measurement range typically of interest for bioanalytical applications. Most dilute aqueous solutions of biologicalrelevance have refractive indices in the range from 1.33 to 1.40. Byusing this model, one is able to calculate the dimensions of thecapillary required to make the abrupt intensity change occur inposition/refractive index space at refractive indices above 1.33 and atreflection angles inside our measurement range. The mechanismresponsible for this abrupt change in intensity is, according to themodel, total internal reflection of the rays reflected from the back ofthe capillary, preventing these rays from being scattered to largerreflection angles, thereby causing a sudden decrease in the intensity ofthe light at a given limiting angle. This angle varies uniformly withthe refractive index and can therefore be used as a measure for therefractive index of the liquid.

The modeled interference pattern as function of refractive index for a542 μm ID/673 μm OD/24 um coating capillary is shown in FIG. 16. FIG. 17shows the experimental results from using a 542 μm ID/673 μm OD/24 umcoating capillary. It is seen by comparison to FIG. 16 that the positionof the abrupt change in intensity differs from the model, although thebehavior is quantitatively the same. Both low and medium frequencyvariations as well as the abrupt change in intensity level at highreflection angles are seen. The abrupt change in intensity for thiscapillary occurs in a more relevant interval for dilute aqueoussolutions, than it does for the 100 μm ID/165 μm OD/12 μm coatingcapillary, as indicated by (a).

The experimental and the modeled results show good agreement. The abruptchange in interference pattern is already detectable at the refractiveindex of water and is experimentally accessible well beyond refractiveindices normally considered for aqueous solutions; see point (a) in FIG.16. The discrepancy between the predicted and the measured angles of theabrupt intensity change may be attributed to material parametertolerances of the experimental set up as follows: The uncertainty of thedimensions of this capillary is 12 μm and 25 μm for the inner and outerdiameter, respectively. This gives an uncertainty of 6.9° in the anglepredicted by the model. The uncertainty in refractive index of thepolyimide coating (n=1.5 to 1.8) can cause a further uncertainty in thepredicted angle of 3.3°. In FIG. 17, the position of the abrupt changein intensity is monitored as function of refractive index of sucrosesolutions. The detection limit for refractive index changes achieved byfollowing the position of this change in position is 2.5×10⁻⁴. From theexperiments resulting in FIG. 17, the precision is found to be 2.5×10⁻⁴.Hence an absolute refractive index measurement with accuracy on thislevel on a nanoliter volume can be performed. The main limitations foraccuracy such as temperature control and detector resolution are thesame as conventional BSI. Theoretical limit using this approach istherefore similar to the limit achievable by conventional BSI.

There are at least two possible ways of making absolute measurements ofthe refractive index of liquid in nanoliter probe volumes in a simpleoptical setup. The improved model based on ray tracing has been used toreveal and explain novel features of the interference pattern: An abruptchange in intensity at large reflection angle is clearly present in themodeled system and in the experimental results. It is shown that thisapproach enables an absolute determination of the refractive index inthe range from 1.33 to 1.5 by using capillary tubes of appropriatedimensions. It has been proven that the model based on ray tracing maybe used for describing the BSI phenomenon. The improved ray tracingmodel is capable of explaining all the significant features of the BSIpattern except the stationary high frequency fringes. However, thesefringes have been shown to originate from reflections from the edges ofthe capillary and thereby not being relevant for measuring therefractive index of the liquid within. These improvements of the BSIscheme can contribute significantly to enhance future applicability ofthe methodology for analysis of minute volumes of aqueous solutions.

D. Detection of Chemical Events

The disclosed systems and methods can be used in connection with thedetection and determination of a wide variety of characteristicproperties of a sample. For example, the invention can be used todetermine absolute or relative refractive index (RI) of a sample, forexample a fluid either flowing or static. The disclosed systems andmethods can also be used in connection with detection and determinationof chemical events, for example label-free analysis of hybridizationreactions such as DNA-DNA binding reactions. The disclosed systems andmethods can also be used in bioassays, monitoring enzymatic activity,drug screening, and clinical diagnostics.

In one aspect, the disclosed methods can be performed wherein thecharacteristic to be determined is whether first and second biochemicalfunctional species (i.e., first and second analytes) bind with oneanother, and the step of introducing a sample to be analyzed into thefirst rectangular channel comprise introducing the first biochemicalfunctional species into the channel and then introducing the secondbiochemical functional species into the channel to facilitate a bindingreaction between the first and second biochemical species. For example,the first and second biochemical functional species can be selected fromthe group comprising complimentary strands of DNA, complimentaryproteins, enzyme-substrate pairs, and antibody antigen pairs. That is,in a further aspect, the characteristic to be determined can be alabel-free analysis of a hybridization reaction in the channel. In a yetfurther aspect, the positional shifts in the light bands can correspondto a chemical event occurring in the sample.

Examples of chemical events that can be detected and bioassays conductedwith the disclosed systems and methods include a binding event betweenone or more of antibody-antigen, protein-protein, small molecule-smallmolecule; small molecule-protein, drug-receptor; antibody-cell;protein-cell; oligonucleotide-cell; carbohydrate-cell; cell-cell;enzyme-substrate; protein-DNA; protein-aptamer; DNA-DNA; RNA-RNA;DNA-RNA; protein-RNA; small molecule-nucleic acid; biomolecule-molecularimprint (MIP); biomolecule-protein mimetic; biomolecule-antibodyderivatives (SCFV, Fab, FC, etc.); lectin-carbohydrate; andbiomolecule-carbohydrate.

In one aspect, the disclosed systems and methods can be used inconnection with a step of performing a chromatographic separation or anelectrophoretic separation on the sample prior to the determining thecharacteristic property step.

1. Analytical Detection Events

The invention also finds use as a detector for other chip-scaleanalytical schemes including electrophoresis, μ-HPLC separations andFIA. It is possible to detect molecules important to cellular function,high throughput analysis, and pharmaceutical screening. Theinterferometer can also be used in biochemical assays and to quantifyenvironmental analytes. It is also possible to performmicro-thermometry, the device has the capability of measuring smalltemperature changes (in the 10⁻³° C. range) allowing for cellularrespiration, protein folding, calorimetry, and fundamental chemicalbinding studies to be performed in picoliter volumes. Furthermore, whenusing special surface chemistry to selectively bind solutes, such as DNAoligomers, proteins, or antibodies, without sacrificingspecificity/sensitivity. Use of the device to perform flow sensing,pressure sensing, time resolved enthalpies and perform detection forproducts eluted from focusing techniques such as flow cytometry is alsoviable, as well as ability to monitor label-free reactions and toquantify the interference brought on by fluorescent markers normallyattached to biomolecules.

2. Determination of Kinetic Parameters

In one aspect, a modified “stop-flow” methodology [B. J. Burke, F. E.Regnier, Analytical Chemistry 75, 1786-1791 (Apr. 15, 2003).] wasadopted, enabled by a PDMS microfluidic chip which is configured withtwo sample reservoirs, both connected to equal length channels thatconverge into a single channel with a serpentine mixer made from aseries of connected C shapes followed by a restriction (see FIG. 22).This simple microfluidic network, which allows for sample introductionand rapid mixing of the two interacting species, was fabricated usingstandard photolithography and replica molding techniques [D. C. Duffy,J. C. McDonald, O. J. A. Schueller, G. M. Whitesides, AnalyticalChemistry 70, 4974-4984 (Dec. 1, 1998); G. M. Whitesides, E. Ostuni, S.Takayama, X. Y. Jiang, D. E. Ingber, Annual Review of BiomedicalEngineering 3, 335-373 (2001).]. After the PDMS was cured and peeledfrom the mold, it was oxidized in O₂ plasma for 10 seconds and thenplaced on a 1-mm thick microscope glass slide creating an irreversiblebond between the glass and PDMS. The glass slide was used to seal themicrofluidic channels and allowed for the entire chip assembly to behandled and securely mounted onto a thermoelectricallytemperature-controlled x-y translation stage. A shorter top piece ofglass was also used as a faceplate, offering structural stability tominimize any possible microfluidic channel deformations during thesample introduction step. Nanoliter volumes of samples of each of thetwo binding pairs were aliquoted in the reservoirs at the top of the“Y”, then a slight negative pressure was then applied to the chip exitwell, drawing the two interacting species through the mixer and into thedetection zone. In “stop-flow” experiments, the sample introductionpressure was selected to optimize the flow rate or linear velocity forthe solutes. The need for rapid, complete mixing to produce ahomogeneous solution of the binding pair was balanced with therequirement that the reaction has not proceeded appreciably before flowis stopped and analysis begins. These parameters can change slightly foreach binding pair, with flow rates found to be in the range of 75-120μL/min.

Protein A (PA) binds the FC region of several IgG species, includinghuman and rabbit, with high affinity (KD=5 nM-34.5 nM) [J. J. Langone,Advances in Immunology 32, 157-252 (1982); K. Saha, F. Bender, E.Gizeli, Analytical Chemistry 75, 835-842 (Feb. 15, 2003).] and providesan excellent model system to show molecular interactions can be studiedusing back-scattering interferometry. FIG. 23A shows the results fromthe stop-flow interaction experiment with this well-studied pair, inwhich the time-dependent intrinsic property changes (RI) were measuredby BSI to determine affinity. In this case, a fixed concentration ofProtein A of 2.5 nM was used and sequential experiments withincreasingly larger concentrations of the FC region from IgG (i.e., from10 to 40 nM) were performed. Conditions were such that PA was bufferedat a pH=7.2 with 15 mM Na2HPO4, 50 mM NaCl, 0.1 mM EGTA, and 0.02%sodium azide. All IgG solutions were made using the same buffer as PA.The temperature of the solutions and micro-fluidic chip were heldconstant at 25° C. throughout the entire experiment. The associationreaction was detected in real-time over a span of ˜60 seconds. The shapeof the binding curves changes with concentration of the antibody (IgG)at fixed concentration of the substrate, receptor or antibody, morerapidly reaching the equilibrium point.

The apparent binding affinity can be extracted from the data using asimple model that assumes first order kinetics or single mode binding,and plotting the observed rate _((Kobs)) versus the concentration ofIgG. Least squares analysis of the line generated by this method usingthe kinetics obtained from backscattering interferometry yields a Kd forPA-IgG of 7.91 nM (±1.21). Alternatively a plot of the end-point valuesof phase as determined by BSI for the reaction between PA and IgG as afunction of the concentration of IgG can be used as a second method toevaluate binding affinity of the complex. This plot (FIG. 23B) exhibitsthe hyperbolic shape often seen in enzyme kinetic studies and describedby the law of mass action. Analysis of the steady-state data by Prism™software yields a Kd value of 6.27 nM (±0.47), which correlates wellwith the results obtained from the kinetic analysis and with valuesreported in the literature [K. Saha, F. Bender, E. Gizeli, AnalyticalChemistry 75, 835-842 (Feb. 15, 2003).]. The nominal leveling offobserved at higher concentrations of the ligand in the end point assaycan be attributed to a bulk RI signal which becomes increasingsignificant at higher concentrations of the ligand, IgG. This backgroundsignal contribution is typically small compared to that of the bindingevent.

As a control, the 2.5 nM solution of PA and a 40 nM solution of the FABfragment of IgG were introduced into the microfluidic chip and thereaction progress was monitored. FIG. 23A demonstrates that combining ahigh concentration of the non-complementary strand with the targetresults in a nominal response by BSI. In fact the control shows <1.6% ofthe signal observed at equivalent PA and IgG FC concentrations, whileexhibiting decidedly different kinetics. Even though a bulk propertychange is expected and observed, the magnitude of this contribution issmall.

Interaction assays by BSI also yield the benefits inherently afforded bymicrofluidics. It was possible to perform the entire Protein A—IgGhomogeneous, label-free binding assay in inexpensive, easy tomanufacture PDMS chips with a mere 105×10⁻⁹ g (2.5 pmoles) of Protein Aand just 287×10⁻⁹ g (5.75 picomoles) of the FC fragment of IgG. Underthe best case scenario, a comparable determination by ITC wouldtypically require 300 to a 1,000-fold more mass (ca. 300 μg) of each ofthe reactants.

Calmodulin (CaM), the ubiquitous, calcium-binding protein that can bindto and regulate a multitude of different protein targets, was chosen tofurther demonstrate the utility of BSI for homogeneous, label-freemolecular interaction studies. CaM is found in the cytoplasm, withinorganelles, or associated with the plasma or organelle membranes andaffects many different functions including inflammation, metabolism,apoptosis, muscle contraction, intracellular movement, short-term andlong-term memory, nerve growth, and the immune response (REFS). Uponbinding to Ca²⁺, CaM undergoes a conformational change thought to induceactivity. Once activated by Ca²⁺, CaM binds, among other things, theprotein calcineurin, a skeletal muscle myosin peptide, and smallinhibitor molecules. Ligands ranging in molecular weight from just 40.04g/mole for an ion to 77 kDa for a protein, and spanning three decades inKd (from a few micomolar to tens of nanomolar) provide an array ofligand-substrate interactions to demonstrate the utility of BSI.

FIG. 24 shows that a single methodology, label-free homogeneous assaysby BSI, can be used to quantify: a) CaM-Ca²⁺ interactions, b)interactions between CaM and the small molecule inhibitor,trifluoperazine dihydrochloride (TFP), c) CaM and Calcineurin binding,and d) reaction of CaM with M1 3, a peptide from the sequence ofskeletal muscle myosin light chain kinase (skMLCK), a known target ofthe Ca²⁺ activated CaM complex.

Calmodulin-calcium ion interaction has been studied previously withpublished Kd values ranging from 1-10 μM. Homogeneous, label-freeCaM-Ca²⁺ interactions are quantified with BSI by real-time monitoringthe sequential reactions of a constant concentration of 5 μM CaM withCa²⁺ solutions increasing in concentrations from 12.5-100 μM (FIG. 24A).Here CaM was buffered at pH=7.5 with 0.1M HEPES and 0.1M KCl, with itsconcentration held constant at 5 μM. In this aspect, the CaM solutioncontained a small amount of EGTA to chelate any free Ca²⁺. The reactionswere carried out in the same microfluidic chip molded in PDMS with thetemperature fixed at 25° C. A 5 μM CaM solution and a 100 μM Ca²⁺solution both consisting of excess EGTA (i.e., 400 μM), served as thecontrol. As shown in FIG. 24A, when evaluated by stop-flow the controlgenerated a nominal response in BSI, showing <3% change in signal abovethat observed in the absence of excess EGTA at equivalent CaM and Ca²⁺concentrations.

Kinetic analysis using a single exponential gives rise to a plot ofobserved rates versus Ca²⁺ concentration that is linear. A least squaresanalysis of the linear plot yields the slope, intercept, and theirrespective errors. From this analysis, Kd was determined to be 3.36 μM(±5.75), which agrees well with the published range of 1-10 μM. Analysisof the plot of the steady-state values monitored by BSI versus theconcentration of Ca²⁺ using Prism™ software yields a Kd value of 17.77μM (±1.55). Without wishing to be bound by theory, it is believed thatthe disparity in these two values was due likely to a less than optimalapproach to fitting the kinetic data and the RI background present fromunreacted ligand, particularly at high concentrations.

Investigations into the interaction between CaM and the small moleculeinhibitor, trifluoperazine dihydrochloride (TFP) have been previouslyexamined using affinity chromatography(42), a lengthy and substrateconsuming technique 30 μg pr sample, each sample is 300 μL. In thesechromatography studies, dissociation constants or affinities ranged from4.5-5.8 μM. Here, BSI was used to quantify the CaM-TFP interactionreadily, rapidly and with only micrograms of sample.

CaM was buffered at pH=7.5 with 0.1M HEPES and 0.1M KCl, itsconcentration was held constant at 2 μM and to ensure CaM was in itsactive conformational state, the solution contained 0.2 mM CaCl2. TFPsolutions were made using the same buffer and held at the same pH asCaM. Throughout the experiment, the temperature was kept constant at 25° C., and the same PDMS microfluidic chip was used. The interaction ofCaM with TFP was monitored by BSI in real-time within a probe volume onthe order of picoliters (FIG. 24B). A 2 μM solution of CaM and a 25 μMsolution of TFP, both in the absence of Ca²⁺, were mixed to serve as acontrol. In this case the control showed <4% of the signal observed atequivalent CaM and TFP concentrations when Ca²⁺ was present.

Kinetic analysis produces a linear relationship between the observedrates over the concentration range of TFP used, and from the leastsquares analysis, KD was determined to be 4.73 μM (±1.07) for theCaM-TFP complex. This value is in excellent agreement with valueobtained by affinity chromatography. A plot of the BSI signal values atsteady-state versus TFP concentration was constructed and fitted usingPrism™ software giving a Kd of 7.64 μM (±0.85).

Calcineurin is a protein phosphatase and the major CaM binding proteinin the brain. The pair has been studied previously using both affinitychromatography and radioligand binding, with dissociation constantsbeing reported between 4 nM and 1 6 nM. The study of this pairillustrates the importance of BSI for chemical interaction studies; nolabeling is required, inherently short analysis times are possible, andagain microfluidics facilitate assays with small amounts of reactants.In this case CaM was buffered at pH=7.5 with 0.1M HEPES and 0.1M KCl and0.2 mM CaCl2, with the CaM concentration held constant throughout theexperiment at 10 nM. FIG. 24C shows that the time-dependent reaction ofCaM with various concentrations of Calcineurin can be recorded with BSI.A 10 nM solution of CaM and a 100 nM solution of Calcineurin both in theabsence of Ca²⁺ were mixed to serve as a control. The control showed<1.5% of the signal observed at the equivalent CaM and Calcineurinconcentrations in the presence of Ca²⁺.

Kinetic analysis yields a linear relationship between the observed ratesand Calcineurin concentration. The slope and intercept from a leastsquares analysis yields a Kd of 15.67 nM (±5.12). The determinationfalls within the results published earlier using affinity chromatographyand radioligand binding [C. B. Klee, M. H. Krinks, Biochemistry 17,120-126 (1978); M. G. of Sciences of the Speaker, S. J. Orlow, T. W.Sturgill, O. M. Rosen, Proceedings of the National Academy United Statesof America-Biological Sciences 80, 329-333 (1983); M. J. Hubbard, C. B.Klee, Journal of Biological Chemistry 262, 15062-15070 (Nov. 5, 1987).].Steady-state or end-point analysis yields a hyperbolic relationship thatwhen analyzed produces a Kd value of 11.57 nM (±0.79).

M1 3, a peptide from the sequence of skeletal muscle myosin light chainkinase (sk-MLCK), is also a known target of the Ca²⁺ activated CaMcomplex and has been shown by Surface Plasmon Resonance (SPR) to bindwith high affinity (KD=1.9 nM-5.5 nM)(46). While SPR has been used inconjunction with microfluidics and can be employed for small volumes atlow concentrations, it relies on immobilization chemistries forattachment of one reactant onto a metal (typically Au) surface. Surfaceimmobilization chemistry can be costly, time consuming, incompatiblewith some materials, and often exhibit decreased activity over time [R.L. Rich, Y. S. N. Day, T. A. Morton, D. G. Myszka, AnalyticalBiochemistry 296, 197-207 (Sep. 15, 2001).]. Immobilization of thereactant to the surface can perturb the species possibly skewing kineticand thermodynamic results. Furthermore the SPR signal falls off rapidlywith the distance from the surface limiting the size of the target andnegating the potential to gain information about bulk solution bindingproperties.

For free-solution BSI studies of the interaction between CaM and M13,CaM buffered at pH=7.5 with 0.1M HEPES, 0.1M KCl, and 0.2 mM CaCl2. Theconcentration of CaM was kept constant throughout the experiment at 5nM. A buffer-matched range of concentrations of M1 3 were reacted withCaM sequentially and time-dependent association events were detected byinterferometry (FIG. 24D). A 5 nM solution of CaM and a 50 nM solutionof M13, both devoid of Ca²⁺, were mixed to serve as a control. Thecontrol showed <2.6% of the signal observed at equivalent CaM and M1 3concentrations when Ca²⁺ was present. Similar to other CaM bindingevents studied, a linear relationship between the observed rates and theligand concentration (M13) enabled the calculation of KD. For theCaM-M13 pair this value was determined to be 2.72 nM (±0.4 1) andcompares well with results published using SPR [S. Montigiani, G. Neri,P. Neri, D. Neri, Journal of Molecular Biology 258, 6-13 (Apr. 26,1996).]. Analysis using the end-point CaM-M13 signal values versusconcentration and Prism™ software yields a KD value of 11.13 nM (±1.21).The increased noise observed in this determination and the larger RIchanges at higher concentration of ligand are the likely causes for thedisparity between the end-point and kinetic affinity values.

Kinetic parameters can also be derived from BSI. For example theinteraction of Calmodulin with M1 3, the peptide sequence from theCalmodulin-binding domain of myosin light chain kinase (MLCK) showremarkable consistency with stopped-flow kinetics performedpreviously(48). The association rate determined by BSI was 3.1×107 M⁻¹s⁻¹ compared to 3.9×107 M⁻¹ s⁻¹ as determined by Török [K. Török,Biochemical Society Transactions 30, 55-61 (April, 2002).].

Homogeneous assays based on calorimetry are typically problematic forvery low and very high binding affinities. To effectively evaluate theinteraction between pairs with picomolar binding affinities, it isdesirable to perform the determination at sub-nanomolar concentrations,which is often not possible with ITC. Due to the sensitivity resultingfrom a multi-pass interferometric optical train, such measurements arepossible with BSI. To demonstrate this unique feature, the interactionbetween IL-2 and a monoclonal antibody was measured in buffer and incell-free media (FIG. 25). Interleukin-2 (IL-2) is a well-studiedprotein [J. Theze, P. M. Alzari, J. Bertoglio, Immunology Today 17, 481-486 (October, 1996); A. K. Abbas, A. H. Lichtman, Cellular andMolecular Immunology (Saunders, Philadelphia, ed. Fifth, 2003).] that issecreted by activated T-cells and is involved in the regulation of theimmune response. IL-2 is responsible for the proliferation ofantigen-specific cells as well as promoting the proliferation anddifferentiation of other immune cells. IL-2 also aids in regulating theapoptotic pathway of antigen-activated T-cells. The interaction betweenIL-2 and its antibody (IL-Ab) have previously been shown to bind withhigh affinity, with reported Kd values ranging from 10 pM to 60 pM [G.H. Reem, N. H. Yeh, D. L. Urdal, P. L. Kilian, J. J. Farrar, Proceedingsof the National Academy of Sciences of the United States of America 82,8663-8666 (December, 1985).].

BSI was used to examine this system in cell media label-free in ahomogeneous format allowing interactions of IL-Ab (2 nM) with IL-2(10-100 pM) to be monitored in real-time. Both the IL-2 and IL-Absolutions were made utilizing RPMI 1640 cell media with 1% fetal bovineserum (FBS) and 10 μg/mL Cipro. A blank (0 M of both IL-2 and IL-Ab) aswell as two controls (0 M IL-2 reacted with 2·10⁻⁹ M IL-Ab; 1·10⁻¹¹MIL-2 mixed on chip with 0 M IL-Ab) were evaluated. A slight RI changewas seen in all the blanks, but the change was consistent for all three,indicating that this was an effect of the mixing/media bulk RI changes.The kinetic analysis, as described above for the CaM and PA-IgG pairs,was performed on the data and yields a linear plot. From the analysis, aKd of 51.8 pM (±10.5 pM) was determined and falls within the publishedrange of 10 pM to 60 pM [G. H. Reem, N. H. Yeh, D. L. Urdal, P. L.Kilian, J. J. Farrar, Proceedings of the National Academy of Sciences ofthe United States of America 82, 8663-8666 (December, 1985).].

E. Molecular Interactions and Biosensor Applications

Molecular interaction analysis is an active area of biomedical researchas scientists look for understanding of which molecules bind to othermolecules. This information can be critical on any number of levels,especially as it pertains to an understanding of the mechanism of actionof pharmaceutical small molecules or biological macromolecules. Thestudy of interactions can also elucidate possible mechanisms of toxicityand can help identify how best to modify molecules to become moreeffective therapeutics. A thorough understanding of which molecules bindwhich molecules can also lead to a more comprehensive understanding ofthe molecular pathways involved in gene function which can help identifynew points of intervention in disease states such as cancer or diabetes,or new points of intervention in the pathways that contribute to aging.Molecular interactions can also provide a rapid diagnostic tool for thepresence or absence of molecules that are correlated with disease orwith the presence of pathogens in the environment.

Historically, scientists have used semi-quantitative methods such asgenetic, biochemical, and structure-function methods that have producedqualitative or semiquantitative interaction data. Beginning in 1990,Biacore introduced the first commercial machine to use surface plasmonresonance (SPR) to study the real time kinetics of biomolecularinteractions. Systems biology approaches will require these types ofdata to better model the huge number of interactions forming specificmolecular networks.

Biosensors have been defined as any type of device that contains abioreceptor and a transducer. The bioreceptor can be a biologicalmolecular species such as a nucleic acid, a protein, enzyme, antibody oreven a living biological system such as cells or whole organisms thatwould bind the target species. The transducer would then convert thisbinding event into a measurement that could be recorded or displayed.Several types of transducers have been developed, including opticalmeasurements (including fluorescence, luminescence, absorption,phosphorescence, Raman, SERS, surface Plasmon resonance, andback-scattering interferometry), electrochemical, and mass-sensitive(including surface acoustic wave and microbalance).

1. Antibody Biosensors

In conventional antibody biosensors, the antibody bioreceptors bind thetarget of interest and then are visualized by binding a secondaryantibody labeled with radioisotopes or conjugated to an enzyme such ashorseradish peroxidase that catalyzes a chemiluminescence reaction thatcan be visualized with photographic film or appropriate photometricsensor. In one aspect, the invention relates to an antibody biosensorbecause BSI in the absence of a secondary antibody can detect theprimary antibody binding the target due to a change in the refractiveindex due to the binding event, for example due to a change inpolarizability of the target.

Accordingly, in a further aspect, the invention relates to a method formethod for free-solution determination of molecular interactionscomprising the steps of providing a substrate having a channel formedtherein for reception of a fluid sample to be analyzed; introducing afirst sample comprising a first non-immobilized analyte to be analyzedinto the channel; introducing a second sample comprising a secondnon-immobilized analyte to be analyzed into the channel; allowing thefirst analyte to interact with the second analyte to form one or moreinteraction products; directing a coherent light beam onto the substratesuch that the light beam is incident on the channel to generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; detectingpositional shifts in the light bands; and determining the formation ofthe one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to detect a targetof interest in the absence of a second antibody.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M, wherein the method is employed to detect a targetof interest in the absence of a second antibody.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL, wherein the method isemployed to detect a target of interest in the absence of a secondantibody.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to detect a targetof interest in the absence of a second antibody.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the system is employed as an antibody biosensor.

2. Nucleic Acid Biosensors

In conventional nucleic acid biosensors, the specific sequence of basesthat define a segment of DNA can be used as a probe to bind other DNAsequences, and these DNA sequences can be labeled with radioactive orother labels. In one aspect, the invention relates to a DNA biosensorbecause BSI in the absence of a labeled secondary DNA probe can detectthe primary DNA binding the target DNA due to a change in the refractiveindex due to the binding event.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns,wherein the method is employed to detect a DNA sequence of interest inthe absence of a labeled secondary DNA probe.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M, wherein the method is employed to detect a DNAsequence of interest in the absence of a labeled secondary DNA probe.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL, wherein the method isemployed to detect a DNA sequence of interest in the absence of alabeled secondary DNA probe.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to detect a DNAsequence of interest in the absence of a labeled secondary DNA probe.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, employed as nucleic acid biosensor.

3. Enzyme Biosensors

In conventional enzyme biosensors, the presence or absence of substratemolecules can be determined by measuring the production of the enzymaticreaction end products. In one aspect, the invention relates to an enzymebiosensor because BSI can be used to measure the amount of the initialsubstrate or the enzymatic reaction end products as long as they arebinding a molecular species where the binding can be detected by achange in the refractive index of the solution. One example can be whenglucose is determined to be present by its binding to a glucose bindingprotein (GBP), an E. coli periplasmic binding protein, wherein theconformation of the GBP changes upon binding the glucose molecule. Incontrast, conventional glucose biosensors, such as the one sold bySenseOmics Inc., utilize a recombinant GBP that has been specificallymodified to include a cysteine residue to which a fluorescent probe isthen attached, and upon binding glucose the conformational change leadsto a decrease in fluorescence intensity.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns,wherein the method is employed to measure the production of theenzymatic reaction end products in the absence of specifically modifiedrecombinant GBP including a fluorescent probe.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M, wherein the method is employed to measure theproduction of the enzymatic reaction end products in the absence ofspecifically modified recombinant GBP including a fluorescent probe.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL, wherein the method isemployed to measure the production of the enzymatic reaction endproducts in the absence of specifically modified recombinant GBPincluding a fluorescent probe.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to measure theproduction of the enzymatic reaction end products in the absence ofspecifically modified recombinant GBP including a fluorescent probe.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the system is employed as an enzyme biosensor.

4. Cellular Biosensors

In conventional cellular biosensors, the presence or absence ofsubstrate molecules can be measured by measuring cellular metabolism,cell respiration, or bacterial bioluminescence. In one aspect, theinvention relates to a cellular biosensor because BSI can be used tomeasure the amount of the initial substrate as long as it is binding amolecular species where the binding can be detected by a change in therefractive index of the solution. One example can be when heavy metalssuch as mercury are determined to be present by their binding to theMerR (metalloregulatory) proteins, wherein the conformation of the MerRproteins changes upon binding the mercury metal ion. In contrast,conventional heavy metal biosensors utilize a recombinant bacterialstrain that has been genetically modified to include a lux reportergene, and then toxicity as a result of the presence of heavy metals canbe indirectly assessed by the diminution of the light signal.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns,wherein the method is employed to directly assay an analyte of interestin the absence of genetically engineered bacteria.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M, wherein the method is employed to directly assayan analyte of interest in the absence of genetically engineeredbacteria.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL, wherein the method isemployed to directly assay an analyte of interest in the absence ofgenetically engineered bacteria.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to directly assayan analyte of interest in the absence of genetically engineeredbacteria.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the system is employed as a cellular biosensor.

5. Measurement of End-Point Values

In one aspect, BSI can measure end-point values of phase for thereaction between molecule A and molecule B as a function of theconcentration of molecule B to determine the binding affinity of thecomplex and/or to quantitatively determine the concentration of the A-Bproduct at reaction equilibrium. End-point concentration bioassays canbe used in both research and clinical diagnostic applications.

In one aspect, the disclosed methods and systems can be used to performsemi-quantitative end-point measurements. A calibration curve for afirst analyte (e.g., antibody) at a known concentration can be generatedby measuring the response to interaction between the first analyte and asecond analyte (e.g., antigen) at systematically varied knownconcentrations. Comparison of a subsequent response measured when anunknown concentration of the second analyte is allowed to interact(e.g., antibody-antigen binding) with a known concentration of the firstanalyte to the calibration curve yields the concentration of the secondanalyte in the sample analyzed. Amount of the second analyte in thesample can then be determined as a function of sample volume.

Thus, in one aspect, the end point of the interaction between a firstnon-immobilized analyte and a second non-immobilized analyte can bedetermined by a method for free-solution determination of molecularinteractions comprising the steps of providing a substrate having achannel formed therein for reception of a fluid sample to be analyzed;introducing a first sample comprising a first non-immobilized analyte tobe analyzed into the channel; introducing a second sample comprising asecond non-immobilized analyte to be analyzed into the channel; allowingthe first analyte to interact with the second analyte to form one ormore interaction products; directing a coherent light beam onto thesubstrate such that the light beam is incident on the channel togenerate backscattered light through reflective and refractiveinteraction of the light beam with a substrate/channel interface and thesample, the backscattered light comprising interference fringe patternsincluding a plurality of spaced light bands whose positions shift inresponse to changes in the refractive index of the fluid sample;detecting positional shifts in the light bands; and determining theformation of the one or more interaction products of the first analytewith the second analyte from the positional shifts of the light bands inthe interference patterns.

In one aspect, the end point of the interaction between a firstnon-immobilized analyte and a second non-immobilized analyte can bedetermined by a method for real-time, free-solution determination ofmolecular interactions comprising the step of detecting the formation ofone or more interaction products of two unlabeled, non-immobilizedanalytes, wherein at least one of the analytes is present during thedetermination at a concentration of less than about 5.0×10⁻⁵M.

In one aspect, the end point of the interaction between a firstnon-immobilized analyte and a second non-immobilized analyte can bedetermined by a method for real-time, free-solution determination ofmolecular interactions comprising the step of detecting the formation ofone or more interaction products of two unlabeled, non-immobilizedanalytes, wherein at least one of the analytes is present during thedetermination in a solution with a volume in the detection zone of lessthan about 500 nL.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein at least one of the analytes is presentduring the determination in a solution with a volume in the detectionzone of less than about 500 nL.

In one aspect, the end point of the interaction between a firstnon-immobilized analyte and a second non-immobilized analyte can bedetermined using an interferometric detection system comprising asubstrate; a channel formed in the substrate for reception of a fluidsample to be analyzed; means for introducing a first sample comprising afirst analyte; means for introducing a second sample comprising a secondanalyte; optionally, means for mixing the first sample and the secondsample; a coherent light source for generating a coherent light beam,the light source being positioned to direct the light beam onto thesubstrate such that the light beam is incident on the channel to therebygenerate backscattered light through reflective and refractiveinteraction of the light beam with a substrate/channel interface and thesample, the backscattered light comprising interference fringe patternsincluding a plurality of spaced light bands whose positions shift inresponse to changes in the refractive index of the fluid sample; aphotodetector for receiving the backscattered light and generating oneor more intensity signals that vary as a function of positional shiftsof the light bands; and a signal analyzer for receiving the intensitysignals, and determining therefrom, a characteristic property of thefluid sample in the channel.

In a further aspect, the first and second analytes can be combined priorto introduction. That is, the analytes can be combined (and thus canpotentially interact) prior to performing the disclosed methods. In thisaspect, the step of introducing the first analyte and the step ofintroducing the second analyte are performed simultaneously.

Alternatively, in a further aspect, the first and second analytes arecombined after introduction. That is, the analytes can be combined at apoint before the channel, or at a point within the channel, whenperforming the disclosed methods. In this aspect, the step ofintroducing the first analyte and the step of introducing the secondanalyte are performed either simultaneously or sequentially. In afurther aspect, the detecting step is performed during the interactionof the first analyte with the second analyte.

6. Determination of Kinetic Parameters

In a further aspect, BSI can determine kinetic parameters. That is, theback-scattering interferometry (BSI) technique described herein can beused to monitor various kinetic parameters, such as, for example,binding affinities, of a chemical and/or biochemical analyte species.The use of BSI for the determination of a kinetic parameter can provideone or more advantages over traditional techniques, for example,free-solution measurements of label-free species, high throughput, smallsample volume, high sensitivity, and broad dynamic range. A BSItechnique can be performed on a free-solution species, a surfaceimmobilized species, or a combination thereof. In one aspect, thespecies of interest is a free-solution species, wherein at least aportion of the species of interest is not bound or otherwiseimmobilized. In another aspect, at least a portion of the species ofinterest is surface immobilized.

In one aspect, a BSI technique can be used to analyze and/or quantifyone or more molecular interactions, such as, for example, a dissociationconstant for one or more binding pair species. Such a binding pairspecies can be, in various aspects, a protein-protein, peptide-protein,small molecule-protein, ion-protein, or an antibody-antigen pair. Otherreactions and/or molecular interactions can be likewise analyzed via BSIand the present invention is not intended to be limited to the specificbinding pairs and/or reactions recited herein.

The sensitivity of a BSI technique can allow analysis and/ordetermination of at least one kinetic parameter to be performed on asmall volume sample. The volume of a sample comprising at least onespecies of interest can, in various aspects, be less than about 1 nL,for example, about 900, 850, 800, 700, 600, 500, 400, 350, 300, 250, or200 pL; less than about 600 pL, for example, about 580, 550, 500, 450,400, 350, 300, 250, or 200 pL; or less than about 400 pL, for example,about 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 280, 250, 230,or 200 pL. In one aspect, the sample volume is about 500 pL. In anotheraspect, the sample volume is about 350 pL. The sample volume can also begreater than or less than the volumes described above, depending on theconcentration of a species of interest and the design of a particularBSI apparatus. A species that can be analyzed via BSI can be present inneat form, in diluted form, such as, for example, in a dilute solution,or any other form suitable for analysis by a BSI technique. Theconcentration of a species of interest can likewise vary depending upon,for example, the design of a particular BSI apparatus, the volume ofsample in the optical path, the intensity of a response of a specificspecies to the radiation used in the experiment. In various aspects, thespecies can be present at a concentration of from about 1 pM to greaterthan 100 mM.

Analysis of a kinetic parameter via a BSI technique can be performed ona static sample, a flowing sample, for example, 75-120 μL/min, or acombination thereof. In one aspect, an analysis can be a stop-flowdetermination that can allow an estimation of the dissociation constant(K_(D)) of one or more binding pairs of species. The speed at which oneor more samples can be analyzed can be dependent upon, inter alia, thedata acquisition and/or processing speed of the detector element and/orprocessing electronics. Methods for adjusting the throughput speed of aBSI apparatus, such as signal multiplexing, can be utilized and areconsidered to be included in various aspects of the present invention.

An apparatus for analyzing a kinetic parameter using a BSI technique cancomprise an optical system and a sample comprising the one or morespecies of interest. The optical system can comprise, a laser, such as,for example, a He—Ne laser, and a detector, such as, for example, a CCDarray detector, such as a high resolution linear CCD. In one aspect, thedetector is a CCD bar code scanner. The sample can be positioned in oron a channel, such as, for example, a microfluidic channel on apoly(dimethylsiloxane) chip. A microfluidic channel, if present, cancomprise a pattern, such as, for example, a serpentine flow pattern,and/or a mixing zone, such as, for example, a squeeze. In a specificaspect, the sample can be positioned in a rectangular channelapproximately 50 μm by 70 μm. In such a specific aspect, the sample canbe irradiated with a 100 μm diameter He—Ne laser beam to yield anoptical sample volume of approximately 350 pL. In other various aspects,a microfluidic channel, if present, can be semicircular or cylindrical,such as, for example, a fused silica capillary, and the presentinvention is not intended to be limited to any particular microfluidicchannel geometry.

A microfluidic channel, if present, can comprise one or multiplechannels that can hold and/or transport the same or varying samples, anda mixing zone. The design of a mixing zone can allow at least initialmixing of, for example, one or more binding pair species. The at leastinitially mixed sample can then be subjected to a stop-flow analysis,provided that the reaction and/or interaction between the binding pairspecies continues or is not complete at the time of analysis. Thespecific design of a microfluidic channel, mixing zone, and theconditions of mixing can vary, depending on such factors as, forexample, the concentration, response, and volume of a sample and/orspecies.

The concentration of one or more analyte species in a sample can bedetermined with a BSI technique by, for example, monitoring therefractive index of a sample solution comprising an analyte species. Aproperty, such as, for example, refractive index, can be measured inreal-time and the kinetics of an interaction between analyte speciesdetermined therefrom. Other experimental conditions, such as, forexample, temperature and pH, can optionally be controlled duringanalysis. The number of real-time data points acquired for determinationof a kinetic parameter can vary based on, for example, the acquisitionrate and the desired precision of a resulting kinetic parameter. Thelength of time of a specific experiment should be sufficient to allowacquisition of at least the minimal number of data points to calculateand/or determine a kinetic parameter. In one aspect, an experiment canbe performed in about 60 seconds.

An apparent binding affinity between binding pair species cansubsequently be extracted from the acquired data using conventionalkinetics models and/or calculations. In one aspect, a model assumesfirst order kinetics (a single mode binding) and the observed rate(k_(obs)) can be plotted versus the concentration of one of the species.A desired kinetic parameter, such as, for example, K_(D), can bedetermined by, for example, a least squares analysis of the relationshipplotted above. A suitable fitting model can be selected based on theparticular experimental condition such that a rate approximation can bedetermined at the end of the analysis. One of skill in the art canreadily select an appropriate model or calculation to determine aparticular kinetic parameter from data obtained via BSI analysis.

7. Immobilized Bait Measurements

In a further aspect, BSI can measure immobilized bait measurements. Oneexample of a measurement of an immobilized bait using BSI is wherebiotin was determined to bind surface-immobilized streptavidin (2004JACS Markov et al. 126:16659-64). The bait can be one of two interactingspecies, and, in conventional immobilized bait measurements, the bait isimmobilized at a surface of the analysis system, wherein the interactionand analysis occur.

In contrast, the disclosed methods and systems can involve free-solutionmeasurements. The disclosed methods and systems, however, can be used tointerrogate analytes that are non-immobilized, yet bound. That is, theanalyte can be selectively bound to another species that, too, is notimmobilized at a surface of the analysis system. More specifically, thebait can be bound upon a substrate that is introduced as a free solutionwith respect to the detection zone. For example, the bait could beimmobilized in a micelle, upon a nanoparticle, or within a cell membranefragment. As further examples, the disclosed systems and methods can beapplied to molecules embedded in micelles, cell membrane segments,intact cells, and/or nanoparticles with derivatized surfaces.

Analogously, one binding partner can be bound upon a nano- ormicrosupport that can then be analyzed within the disclosed systemsunder free solution conditions by using the disclosed methods.

As a further example, an analyte can be bound to a magnetic particle anddelivered (or held) by magnetic fields at a desired detection zonewithin a larger system (e.g., a biological system such as an organism).

In a further example, one or more of the interacting analytes can bebound by “tether” to a surface of the system within the detection zone.Another, free-solution analyte can the be allowed to interact with thebound analyte, thereby forming one or more interaction products, whichcan remain bound via the tether or can be released into free solution.The tether can be, for example, attachment via covalent bond or otherstrong interaction. The attachment directly to the surface, attachmentvia a relatively short tether (e.g., functionalized alkyl chain,oligomer, or self-assembled monolayer) or via a long tether (e.g.,functionalized alkyl chain or polymer—potentially hundreds or thousandsof nanometers in length).

In conventional techniques, a first analyte is attached to a surface(e.g., via tether attachment). The attachment is typically followed by awash step to remove remaining unattached first analyte from the sampleor detection zone; this wash can be necessary to eliminate or minimizeany interaction due to non-immobilized first analyte. The wash step isfollowed by the addition of a second analyte, which interacts with theimmobilized first analyte to form one or more immobilized interactionproducts. In conventional techniques, this step is followed by a secondwash step to remove remaining non-interacted second analyte from thesample or detection zone; this wash can be necessary to eliminate orminimize any interaction due to non-interacted second analyte. Incontrast, in the disclosed methods and systems—at least in part due tothe sensitivity of BSI when observing interaction products frominteracting analytes—the second wash step can be unnecessary, as thesignal observed in response to the formation and presence of the one ormore interaction products is strong relative to any response dueunreacted analytes. Thus, the disclosed methods can be performed for theanalysis of interaction between an immobilized or tethered analyte and anon-immobilized analyte while omitting the second wash step.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns,wherein the method is employed to investigate non-immobilized, yet boundanalytes.

Accordingly, in a further aspect, the invention relates to a method forreal-time, free-solution determination of molecular interactionscomprising the step of detecting the formation of one or moreinteraction products of two unlabeled, non-immobilized analytes, whereinat least one of the analytes is present during the determination at aconcentration of less than about 5.0×10⁻⁵M, wherein the method isemployed to investigate non-immobilized, yet bound analytes.

Accordingly, in a further aspect, the invention relates to a method forreal-time, free-solution determination of molecular interactionscomprising the step of detecting the formation of one or moreinteraction products of two unlabeled, non-immobilized analytes, whereinat least one of the analytes is present during the determination in asolution with a volume in the detection zone of less than about 500 nL,wherein the method is employed to investigate non-immobilized, yet boundanalytes.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to investigatenon-immobilized, yet bound analytes.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the method is employed to investigatenon-immobilized, yet bound analytes.

8. Free Solution Measurements

In a further aspect, BSI can measure free solution measurements. Oneexample of a free solution measurement in life science applications canbe when the BSI instrument is used to interrogate the binding of twobiological macromolecules, such as IL-2 and a monoclonal antibody forIL-2, in solution by examining a change in the interference patternproduced from the reflection and refraction of the solution upon mixingthe two biological macromolecules. In contrast, conventional methodsrequire measuring the amount of IL-2 bound with monoclonal antibody for11-2 by for example Western blotting that requires tethering the IL-2 toa solid support, binding the antibody, and then binding a secondaryantibody that has a label attached to it for visualization. In contrast,the BSI method does not require that the protein being examined be boundto a solid support, as the measurement could be made in free solution.

Other surface-bound biosensor techniques can be supplanted by thedisclosed free-solution methods and systems. For example, the objectiveof surface plasmon resonance (SPR), optical wave-guide techniques,grating coupled optical waveguide techniques, micro-cantilevertechniques, atomic force microscopy, acoustic techniques, as well aslabeled techniques (including chemiluminescence, ELISA, fluorescencedetection, and solid or liquid scintillation) can be achieved with thedisclosed systems and methods.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the method is employed to measure one or morecharacteristic properties and/or chemical events in free solution (i.e.,non-immobilized analytes).

9. Label-Free Molecular Interactions

In a further aspect, BSI can measure label-free molecular interactions.One example of a label-free measurement in life science applications canbe when the BSI instrument is used to interrogate the binding of twobiological macromolecules, such as a DNA binding protein and thefragment of DNA that contains the sequence that the protein binds byexamining a change in the interference pattern produced from thereflection and refraction of the solution upon mixing the two biologicalmacromolecules. In contrast, conventional methods require DNAoligonucleotides to be immobilized prior to measuring the binding of asingle-stranded DNA binding protein which was visualized using surfaceplasmon resonance (1999 JACS Brockman et al., 121:8044-51). In contrast,the BSI method does not require that the protein being examined belabeled or be bound to a solid support, as the measurement could be madein free solution

In a further aspect, BSI can measure classes of biomolecular interactionstudies as described herein. As used herein, proteins includesglycoproteins, lectins, peptides, antibodies, protein antibody mimeticand any antibody subclasses including SCFV, Fab, Fc, or molecularimprints (MIP). In a further aspect of the invention, the biomolecularinteraction is an interaction of a protein with a protein. In a furtherof the invention, the biomolecular interaction is an interaction of anantibody with an antigen. In a further aspect of the invention, thebiomolecular interaction is an interaction of an enzyme and a substrate.In a further aspect of the invention, the biomolecular interaction is aninteraction of a protein and a virus. As used herein, virus includesphage. In a further aspect of the invention, the biomolecularinteraction is an interaction of a receptor and a ligand. In a furtheraspect of the invention, the biomolecular interaction is an interactionof a protein and a carbohydrate. In a further aspect of the invention,the biomolecular interaction is an interaction of a protein and anucleic acid. As used herein, nucleic acid includes DNA, RNA, andaptamers. In a further aspect of the invention, the biomolecularinteraction is an interaction of a receptor and a ligand. In a furtheraspect of the invention, the biomolecular interaction is an interactionof a nucleic acid with a nucleic acid. In a further aspect of theinvention, the biomolecular interaction is an interaction of a smallmolecule with a protein. In a further aspect of the invention, thebiomolecular interaction is an interaction of a small molecule with anucleic acid. In a further aspect of the invention, the biomolecularinteraction is an interaction of a small molecule with a receptor. In afurther aspect of the invention, the biomolecular interaction is aninteraction of a small molecule and a carbohydrate. In a further aspectof the invention, the biomolecular interaction is an interaction of asmall molecule and a virus. In a further aspect of the invention, thebiomolecular interaction is an interaction of a small molecule with asmall molecule. In a further aspect of the invention, the biomolecularinteraction is an interaction of a cell with a protein. In a furtheraspect of the invention, the biomolecular interaction is an interactionof a cell with a carbohydrate. In a further aspect of the invention, thebiomolecular interaction is an interaction of a cell with a cell. In afurther aspect of the invention, the biomolecular interaction is aninteraction of a cell with a small molecule. In a further aspect of theinvention, the biomolecular interaction is an interaction of a cell witha nucleic acid. In a further aspect of the invention, the biomolecularinteraction is an interaction of a cell with a virus.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns,wherein the method is employed to measure one or more characteristicproperties and/or chemical events of unlabelled (i.e., substantiallylabel-free) analytes.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M, wherein the method is employed to measure one ormore characteristic properties and/or chemical events of unlabelled(i.e., substantially label-free) analytes.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL, wherein the method isemployed to measure one or more characteristic properties and/orchemical events of unlabelled (i.e., substantially label-free) analytes.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to measure one ormore characteristic properties and/or chemical events of unlabelled(i.e., substantially label-free) analytes.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the system is employed to measure one or morecharacteristic properties and/or chemical events of unlabelled (i.e.,substantially label-free) analytes.

For the detection of biomolecular interactions, the following types ofdetectors can be replaced or can be able to be used in combination withBSI, including optical techniques including Surface enhanced Ramanspectroscopy, and Surface Plasmon Resonance (SPR), SPR is an opticalphenomenon used for measuring molecular interactions but requires thatone molecular species be immobilized. The SPR signal arises in thinmetal films and the signal depends on the refractive index of solutionsin contact with the metal surface. A challenging aspect of using SPR isdirect immobilization of one of the molecular species without disruptingits binding activity. In contrast to SPR, BSI can be used to measure thebinding of macromolecules without either macromolecule being fixed to asurface. For example, using SPR, it was recently shown that solublemonomeric beta-amyloid peptides can bind anti-beta-amyloid monoclonalantibodies (J Phys Chem B 2007; 111: 1238-43). In contrast, BSI can alsobe used to measure soluble monomeric beta-amyloid peptides binding ananti-beta-amyloid monoclonal antibodies in free solution.

A further type of detector that can be replaced or used in combinationwith BSI is one that utilizes grating based approaches such as opticalwaveguide lightmode spectroscopy (OWLS). OWLS measures the surfaceimmobilization of biomolecules in an aqueous solution. The technique isbased on the incoupling of a laser into a waveguide by an opticalgrating. The incoupling only occurs at two defined angles that aresensitive to a change in the refractive index above the surface in theevanescent field. The OWLS method uses the change in the refractiveindex to measure the adsorbed mass. A challenging aspect of using OWLSis direct immobilization of one of the molecular species. In contrast toOWLS, BSI can be used to measure the binding of macromolecules withouteither macromolecule being fixed to a surface. For example, using OWLS,the interaction between mycotoxins and anti-mycotoxin monoclonalantibodies was measured (Biosens Bioelectron 2007 22:797-802). Incontrast, BSI can also be used to measure the binding of solublemycotoxins binding anti-mycotoxin monoclonal antibodies in freesolution.

A further type of detector that can be replaced or used in combinationwith BSI is one that utilizes mass-sensitive measurements such assurface acoustic wave (SAW). In SAW, small mass changes can be measuredthat result from molecules binding the receptor molecules coupled to theactive sensor surface. Small mass changes at the sensor surface affectsthe propagation velocity of acoustic shear waves traveling through aguiding layer at the sensor surface. A challenging aspect of using SAWis direct immobilization of one of the molecular species. In contrast toSAW, BSI can be used to measure the binding of macromolecules withouteither macromolecule being fixed to a surface. For example, using SAW,the interaction between bovine immunoglobulin G and Protein A wasrecently measured (International Conference on Solid State Sensors andActuators Jun. 16-19, 1997 1:187-190). In contrast, BSI can also be usedto measure the binding of bovine immunoglobulin G and Protein A in freesolution.

A further type of detector that can be replaced or used in combinationwith BSI is one that utilizes mass-sensitive measurements utilizing apiezoelectric crystal. For example, small mass changes can be measuredthat result from molecules binding the receptor molecules coupled to theactive sensor surface due to a change in the oscillation frequency of apiezoelectric crystal. Piezoelectric crystals oscillate as a function ofboth the electrical frequency applied to the crystal and the crystal'smass. Small mass changes can therefore be measured electrically. Incontrast to a microbalance, BSI can be used to measure the binding ofmacromolecules without either macromolecule being fixed to a surface.For example, using a piezoelectric crystal, the interaction betweenStaphylococcal Enterotoxin B (SEB) and anti-SEB polyclonal antibodieswas measured (Biosens Bioelectron 1997 12:661-7). In contrast, BSI canalso be used to measure the binding of Staphylococcal Enterotoxin B andanti-SEB polyclonal antibodies in free solution.

10. Electrochemical Measurements

A further type of detector that can be replaced or used in combinationwith BSI is one that utilizes electrochemical measurements. For example,one electrochemical biosensor can detect L-phenylalanine via activity ofthree immobilized enzymes. The three enzymes are immobilized on anelectrode wherein first L-phenylalanine dehydrogenase binds and reactswith L-phenylalanine producing NADH. Then salicylate hydroxylase usesoxygen and NADH to convert salicylate to catechol. Then tyrosinaseoxidizes catechol to o-quinone which is reduced back to catechol with anelectrode potential of −50 mV (Anal Commun 1999 36:281). In contrast tothe electrochemical biosensor, BSI can be used to directly measure thepresence of L-phenylalanine by its binding to another macromolecule infree solution.

Accordingly, in a further aspect, the invention relates to a method forfree-solution determination of molecular interactions comprising thesteps of providing a substrate having a channel formed therein forreception of a fluid sample to be analyzed; introducing a first samplecomprising a first non-immobilized analyte to be analyzed into thechannel; introducing a second sample comprising a second non-immobilizedanalyte to be analyzed into the channel; allowing the first analyte tointeract with the second analyte to form one or more interactionproducts; directing a coherent light beam onto the substrate such thatthe light beam is incident on the channel to generate backscatteredlight through reflective and refractive interaction of the light beamwith a substrate/channel interface and the sample, the backscatteredlight comprising interference fringe patterns including a plurality ofspaced light bands whose positions shift in response to changes in therefractive index of the fluid sample; detecting positional shifts in thelight bands; and determining the formation of the one or moreinteraction products of the first analyte with the second analyte fromthe positional shifts of the light bands in the interference patterns,wherein the method is employed to directly assay an analyte of interestin the absence of one or more specially modified enzymes, or an enzymecascade.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M, wherein the method is employed to directly assayan analyte of interest in the absence of one or more specially modifiedenzymes, or an enzyme cascade.

In a further aspect, the invention relates to a method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination in a solution with a volumein the detection zone of less than about 500 nL, wherein the method isemployed to directly assay an analyte of interest in the absence of oneor more specially modified enzymes, or an enzyme cascade.

In a further aspect, the invention relates to a method for free-solutiondetermination of molecular interactions comprising the steps ofproviding a substrate having a channel formed therein for reception of afluid sample to be analyzed; introducing a first sample comprising afirst non-immobilized analyte to be analyzed into the channel;establishing a baseline interferometric response by directing a coherentlight beam onto the substrate such that the light beam is incident onthe channel to generate backscattered light through reflective andrefractive interaction of the light beam with a substrate/channelinterface and the sample, the backscattered light comprisinginterference fringe patterns including a plurality of spaced light bandswhose positions shift in response to changes in the refractive index ofthe first sample; introducing a second sample comprising a mixture ofthe first non-immobilized analyte and a second non-immobilized analyteto be analyzed, wherein the first analyte to interacts with the secondanalyte to form one or more interaction products, into the channel;directing a coherent light beam onto the substrate such that the lightbeam is incident on the channel to generate backscattered light throughreflective and refractive interaction of the light beam with asubstrate/channel interface and the sample, the backscattered lightcomprising interference fringe patterns including a plurality of spacedlight bands whose positions shift in response to changes in therefractive index of the second sample; detecting positional shifts inthe light bands relative to the baseline; and determining the formationof the one or more interaction products of the first analyte with thesecond analyte from the positional shifts of the light bands in theinterference patterns, wherein the method is employed to directly assayan analyte of interest in the absence of one or more specially modifiedenzymes, or an enzyme cascade.

Moreover, in a further aspect, the invention relates to aninterferometric detection system comprising a substrate; a channelformed in the substrate for reception of a fluid sample to be analyzed;means for introducing a first sample comprising a first analyte; meansfor introducing a second sample comprising a second analyte; optionally,means for mixing the first sample and the second sample; a coherentlight source for generating a coherent light beam, the light sourcebeing positioned to direct the light beam onto the substrate such thatthe light beam is incident on the channel to thereby generatebackscattered light through reflective and refractive interaction of thelight beam with a substrate/channel interface and the sample, thebackscattered light comprising interference fringe patterns including aplurality of spaced light bands whose positions shift in response tochanges in the refractive index of the fluid sample; a photodetector forreceiving the backscattered light and generating one or more intensitysignals that vary as a function of positional shifts of the light bands;and a signal analyzer for receiving the intensity signals, anddetermining therefrom, a characteristic property of the fluid sample inthe channel, wherein the system is employed as an electrochemicalmeasurement device.

11. Atomic Force Microscopy

A further type of detector that can be replaced or used in combinationwith BSI is one that utilizes atomic force microscopy (AFM). AFMutilizes the deflection of a microscale cantilever by forces such aselectrostatic or Van Der Waal etc. in order to scan a specimen at thenanometer scale. The technique can be used to image, measure ormanipulate matter. For example, AFM has been used to measure thedissociation rate constants of aptamer protein complexes (Chem Asian J2007 2:284-9). In contrast to AFM, BSI can be used to measureequilibrium dissociation rate constants of aptamer protein complexes infree solution.

12. End User Applications

BSI can be used in any market where measuring macromolecularinteractions is desired. In basic life science research, betterunderstanding of how proteins interact with one another in the complexnetworks that form biochemical and genetic regulatory pathways can leadto a better understanding of new potential intervention points.

For example, improperly functioning networks, due to inherited orsomatic genetic mutations, can be probed with the disclosed systems andmethods.

Drug discovery and development, as well as translational research, canalso greatly benefit from the disclosed invention, because it offersalternatives for analysis wherein therapeutics bind a target molecule,thereby enabling further development of drug candidates. Modificationsto drug candidates can also be assessed using BSI as a tool to determinebinding properties to the target of interest. Strong and specificbinding can be important for effective therapeutics. Moreover,understandings of which biomarkers are useful for predicting drugefficacy can benefit from tests for their presence in patients, as wellas tests that help elucidate their basic biochemical and physiologicproperties. It is contemplated that the disclosed invention canfacilitate drug discovery, drug development, and translational research.

In the food industry, as well as in biodefense applications, a rapidmethodology that can assay for the presence of toxins, xenobiotics,allergens, additives, or biowarfare agents whether chemicals, viruses,or cellular pathogens such as certain bacteria can be useful asevidenced today by a large number of such items for which no easy to usetests are readily available today. It is contemplated that the disclosedinvention can find utility in food industry and biodefense applications.

The disclosed invention can also be used in clinical diagnostics forearly diagnosis of disease, monitoring disease progression, measurementof drug response to disease, and other applications of personalizedmedicine diagnostics, such as determining optimum drug dosage or drugfor each individual based on diagnostic testing.

F. Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Device Fabrication

A fluidic network was designed using commercially available software(CleWin 2.7). A soda lime/chrome lithographic mask (chrome thicknessapproximately 100 nm) was then prepared (Delta Mask, The Netherlands)using this fluidic network design. Master molds were subsequentlycreated from the lithographic mask using conventional optical and softlithographic techniques.

Three inch silicon wafers (P<100>) were cleaned by sonication in acetonefollowed by treatment with piranha solution. The sonicated and treatedwafers were then rinsed with deionized water and placed on a hot plateat 95° C. for 5 minutes just prior to deposition of a photoresist. Anegative photoresist (SU-8 2050, available from Microchem, Newton,Mass., USA) was then evenly deposited on the surface of the Si waferusing a bench-top single wafer spinner (Laurell WS-400). A fewmilliliters of the negative photoresist were poured onto the center ofthe wafer and spinning commenced for 10 seconds at 500 rpm to spread thephotoresist. The speed of the wafer was then increased to 3000 rpm for40 seconds to form a homogeneous coating. The wafer was then removedfrom the spin coater and placed on a hot plate for a soft bake (3 min at65° C.⇒9 min at 95° C.). The wafer was subsequently allowed to cool toroom temperature. UV exposure through the photolithographic mask for ˜15seconds was accomplished using a Laurell WS-400 Bench-top single waferspinner contact mask aligner. Following irradiation, a post exposurebake (PEB) was performed (1 min at 65° C.⇒7 min at 95° C.). The waferwas again cooled to room temperature.

Unexposed areas of photoresist were then removed using an organicdeveloper (SU-8, available from Microchem). Isopropyl alcohol (IPA) wasused to ensure the wafer was completely developed. IPA will form a milkywhite substance on the wafer if any unexposed photoresist remains. Themaster mold was then rinsed and hard baked (˜5 hours at 2200° C.) toensure device stability. An Alphastep 200 stylus surface profiler(Tencor Instruments) was used to accurately measure the height of thestanding relief structures.

All binding assays were performed in microchips created by cast moldingonto the master mold fabricated above. Cast molding was performed usinga silicon elastomer, polydimethylsiloxane (PDMS), purchased as Sylgard184 (Dow Corning, Midland, Mich.). Prior to casting, the PDMS was mixedin a 10:1 ratio (base:curing agent) and degassed.

PDMS was cast over the master that had been placed into a 100×15 mmFalcon Petri dish (Becton Dickinson, Franklin Lakes, N.J.) such that theheight of the PDMS was ˜2 mm. The Petri dish was placed into adesiccator, and a vacuum was applied for further degassing. Once no airbubbles were visibly present, the Petri dish was removed from thedesiccators and set in a large convection oven for roughly 8 hours at65° C.

After the curing process was complete, the Petri dish was removed fromthe oven and allowed to cool briefly. The PDMS microchip device wasphysically removed from the Si master mold by fine precision scalpel andtweezers. Access ports for sample introduction (2 ports) and appliedvacuum/waste removal (1 port) were mechanically punched out by stainlesssteel capillary tubing. PDMS, with the fluidic network facing up, wasthen plasma oxidized for ˜10 sec along with a 3″×1″×1 mm microscopeglass slide (Fisher Scientific) cleaned in the same fashion as the bareSi wafer. Following oxidation, the PDMS was sealed to the microscopeslide so that the fluidic network was in contact with the glass. Waterwas kept in the channels molded in the PDMS until experiments were runto help maintain the hydrophilic surface created by plasma oxidation.

2. DNA Hybridization

Hybridization of single stranded DNA (ssDNA) to its complimentary strand(cDNA) were performed in 50 μm×50 μm rectangular microfluidic channelsmolded in PDMS in the probe volume of 2.5×10⁻¹° L. The mouse Actin ssDNAsurface immobilization was performed in three steps: first a photoactiveform of biotin was deposited onto channel walls and activated with UVlight; then avidin was introduced into the channel and allowed to reactwith immobilized photobiotin; next injected biotinalated ssDNA wasallowed to react with the immobilized avidin. A 2048-element array incombination with Fourier analysis was used to quantify the positionalchange of the fringe pattern. The change in absolute signal due tohybridization and denaturization is shown in FIG. 20. Using thesingle-channel configuration of OCIBD and when reaction kinetics are notdesired, the signal is recorded in two stages: a) when only ssDNApresent on the channel surface and the fluid within the channel is thePBS buffer; and b) after the introduced cDNA strand has fully reactedwith the immobilized ssDNA strand and after the PBS buffer has beenreintroduced into the channel. This approach allows for the eliminationof erroneous results due to bulk RI changes from the target speciessolution. From the signal magnitude of a determination, the analyticalutility is demonstrated with a simple calculation. Using the parameters:Avidin dimensions of 5.6×5 nm, a probe volume of 2.5×10⁻¹⁰ L, Avogadro'snumber, and based on the worst-case scenario assumption that 100% of thesurface is covered with avidin and 100% of it is reacted with ssDNA1.2×10⁻¹⁶ mol (12 fmol) of bound DNA can be reliably detected. As shownin FIG. 21 this determination gives a result with a relatively largesignal to noise (S/N) ratio. Further interrogation of the data suggeststhe S/N=13, so the 3δ detection limits would be 3 fmol of target DNAreacting with its counter part. These results represent an approximatelytwo-decade improvement over SPR.

3. Interaction of Calmodulin with a Small Molecule Inhibitor

Interactions of Calmodulin (Sigma, St. Louis, Mo.) with a small moleculeinhibitor, a small peptide, and a binding protein were performed in acalcium containing buffer system (i.e., 0.1 M HEPES((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1 M KCl, and0.2 mM CaCl₂ at pH=7.5) while reactions of Calmodulin to Ca²⁺ wereinvestigated using a non-calcium containing buffer (i.e., 0.1 M HEPES,0.1 M KCl, and 0.1 mM ethylene glycol tetraacetic acid (EGTA) atpH=7.5). The pH of buffer solutions was adjusted to the required pH byaddition of 1N HCl or 1N KOH. Solutions were subsequently filtered anddegassed prior to binding experiments. These experiments were conductedat 25±0.01° C. maintained by a thermoelectric temperature controller(MELCOR, Trenton, N.J.) coupled to a Peltier device. Binding experimentswere monitored in real-time at frequencies ca. 50-100 Hz.

A small molecule inhibitor, trifluoperazine dihydrochloride (480 g/mol,Sigma, St. Louis, Mo.), was varied in concentration from 5-25 μM usingthe calcium containing Buffer prepared above. Calmodulin, held constantat 2 μM, and the inhibitor were both introduced on-chip and mixedin-line. CaM's interaction with each concentration of inhibitor wasmonitored and the real-time association observed.

4. Binding Kinetics of Calmodulin and M13 Peptide

The small M13 peptide used was a 17 residue(Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu)peptide from the sequence of myosin light chain kinase. The M13 peptide,(2074.5 g/mol, Calbiochem, La Jolla, Calif.) was aliquoted intoconcentrations ranging from 5-50 nM using the calcium containing buffer.

Calmodulin, held constant at 5 nM, was mixed on-chip with eachconcentration of the M13 peptide. The kinetics of Calmodulin binding M13peptide was monitored and recorded for later analysis.

5. Interaction of CaM and Calcineurin

The CaM and Calcineurin interactions were performed in similarconditions to those of the M13 peptide. CaM was again buffered at apH=7.5 with 0.1M HEPES and 0.1M KCl. CaM's concentration was heldconstant throughout the experiment at 10 nM.

The CaM solution once more contained 0.2 mM CaCl2 to ensure CaM was inits active conformational state. The interaction of CaM with variousconcentrations (10-100 nM) of Calcineurin was monitored by BSI. Allconcentrations of Calcineurin used for the association curves were madeusing the same buffer prepared above and held at the same pH as CaM. Thetemperature was held constant at 25° C. throughout the entireexperiment. A 10 nM solution of CaM and a 100 nM solution of Calcineurinboth in the absence of Ca²⁺ were mixed to serve as a control. Thecontrol showed <1.5% of the signal observed at the equivalent CaM andCalcineurin concentrations when Ca²⁺ was present.

Calmodulin was also reacted to a small metal ion, Ca²⁺. CaM was bufferedat a pH=7.5 with 0.1M HEPES and 0.1M KCl with its concentration heldconstant throughout the experiment at 5 μM. The CaM solution contained asmall amount of EGTA to chelate any free Ca²⁺. BSI monitored, inreal-time, the sequential reactions of 5 μM CaM with concentrations ofCa²⁺ ranging from 12.5-10004.

6. Determination of Binding Affinity

A laser and temperature controller were powered on and allowed toequilibrate over a one hour period prior to starting the experiments. Amirror above a microfluidic chip was positioned so that the incidentlaser beam was directed onto a flow channel orthogonal to fluid flow.The centroid of the backscattered interference pattern was located justabove the focusing lens insuring that the alignment of the system wasalong a central plane. A CCD array was positioned near directbackscatter in order to obtain a high-contrast fringe pattern and adominant Fourier frequency, generally near the 5^(th) or 6^(th) fringefrom the centroid.

Recorded kinetic data was analyzed according to the followingderivation. Since two reactants are mixed on-chip in solution phase BSImolecular interactions, a quantitative solution to a generic,bimolecular reversible reaction was modeled by the analytical solutionof a homogeneous linear first order ordinary differential equation(ODE).

Interactions of CaM and various ligands were analyzed in this manner.Observed rates determined from exponential fits of kinetic traces wereplotted versus the concentrations of the various ligands. The resultingplots exhibited a linear response to concentration with coefficients ofcorrelation ranging from 0.989 to 0.997. Equilibrium dissociationconstants (K_(D)) can be determined from each graph to yield the bindingaffinity of each interaction.

Division of the y-intercept by the slope of each best fit line yieldscan then provide the desired K_(D). The approximation of constant ligandconcentration during a reaction was affirmed from the linearity of eachdata set and a quantitative test of the approximation performed by usingthe rate constants obtained from the linear least-squares results, alongwith the initial concentrations in the experiments, to compare theconcentration of the reaction product at a specific time to the initialconcentration of one of the reactants. Data analysis software can beused to determine K_(D).

7. Free-Solution Label-Free Detection of α-Crystallin ChaperoneInteractions

The binding of various protein targets by the small heat-shock protein(sHSP) α-crystallin was monitored in real time using nanoliter volumesof sample at physiologically relevant concentrations. BSI was used tomonitor the kinetics and analyze the thermodynamics of interactionsbetween α-crystallin and a model substrate, T4 lysozyme (T4L), in alabel-free, tether-free format. Traditional detection methodologiesincluding fluorescence and calorimetry were also used to cross-validateBSI. The specificity of the detection methodology was determined bycomparing the interaction of αB-crystallin with two mutants of T4Lhaving similar structures but different free energies of unfolding.Lastly, a physiologically relevant system was chosen to demonstrate thepotential use of BSI in screening libraries of structural analogs andmutant constructs. Molecular interactions of a mutant α-crystallingenetically linked to autosomal dominant cataract [Mackay, D. S.,Andley, U. P. & Shiels, A. Cell death triggered by a novel mutation inthe alphaA-crystallin gene underlies autosomal dominant cataract linkedto chromosome 21q. Eur J Hum Genet 11, 784-93 (2003)] with another lensprotein, β-crystallin, was analyzed by BSI. Quantitative kinetic andthermodynamic results from BSI were compared to the label-free, surfacesensing technique SPR.

a. Method Comparison and Benchmarking of BSI

The interaction of sHSP with T4L can be represented by a coupled system(FIG. 26). T4L can partially or globally unfold, occupying differentenergy states thus altering the affinity for sHSP binding. sHSPthemselves rapidly exchange subunits and dissociate into different sizedmultimers that have higher affinity for substrate [Shashidharamurthy,R., Koteiche, H. A., Dong, J. & McHaourab, H. S. Mechanism of chaperonefunction in small heat shock proteins: dissociation of the HSP27oligomer is required for recognition and binding of destabilized T4lysozyme. J Biol Chem 280, 5281-9 (2005)]. Furthermore, the binding ofsHSP to T4L has been shown to be bi-modal with sHSP having low and highaffinity sites [Sathish, H. A., Stein, R. A., Yang, G. & McHaourab, H.S. Mechanism of chaperone function in small heat-shock proteins.Fluorescence studies of the conformations of T4 lysozyme bound toalphaB-crystallin. J Biol Chem 278, 44214-21 (2003); Shi, J., Koteiche,H. A., McHaourab, H. S. & Stewart, P. L. Cryoelectron microscopy and EPRanalysis of engineered symmetric and polydisperse Hsp16.5 assembliesreveals determinants of polydispersity and substrate binding. J BiolChem 281, 40420-8 (2006)]. Here, we compare the binding affinitydetermined from steady-state ITC and fluorescence analyses for abimane-labeled T4L mutant and the B subunit of α-crystallin to thatdetermined by BSI.

The αB-crystallin construct used in this study was a triply substitutedanalog of the native protein in which serine residues 19, 45, and 59were mutated to aspartic acids. At physiological pH, the acidic residuescreate a triply charged αB-crystallin species that mimicsphosphorylation. Referred to as αB-D3, this sHSP construct has increasedaffinity for non-native proteins presumably due to a shift in theequilibrium of sHSP dissociation 21 (FIG. 26a ). The substrate,T4L-L99A, is a mutant which has been exhaustively characterized againstmultiple sHSP20-24. With an alanine substituted for leucine at thehydrophobic core position 99, the buried surface area of T4L-L99A isreduced causing enlargement of preexisting cavities [Eriksson, A. E.,Baase, W. A. & Matthews, B. W. Similar hydrophobic replacements of Leu99and Phe153 within the core of T4 lysozyme have different structural andthermodynamic consequences. J Mol Biol 229, 747-69 (1993)]. Thestructure of T4L-L99A is nearly identical to that of the wild-type (WT)T4L with only minor rearrangement in the core. The mutation lowers theGibb's free energy of unfolding (ΔG_(unf)) by 5.1 kcal/mol relative toWT23, 24.

Upon titration with increasing T4L-L99A concentrations to a constantamount of αB-D3 (FIG. 26b ), an increase in the absolute BSI signal wasobserved. A binding isotherm was obtained by plotting the steady-stateamplitude of the BSI signal versus the concentration of T4L-L99A. Thisis equivalent to the fluorescence-based binding isotherms [Sathish, H.A., Stein, R. A., Yang, G. & McHaourab, H. S. Mechanism of chaperonefunction in small heat-shock proteins. Fluorescence studies of theconformations of T4 lysozyme bound to alphaB-crystallin. J Biol Chem278, 44214-21 (2003)]. except that the concentration of T4L is beingvaried rather than sHSP. There are at least two distinguishingcharacteristics of the BSI isotherm. First, the end point values do notappear to reach saturation; rather, they increase linearly at highconcentrations of ligand (♦—FIG. 26b ). Second, a similar pattern wasalso observed with the starting values (▪—FIG. 26b ) for eachαB-D3⋅T4L-L99A binding event. The linear increase in signal at t=0 wasfound to reflect increases in the free T4L concentration. This trend wasnot seen in previous binding assays using BSI as concentrations of thereactants used were small; therefore, changes in the amount of freeligand led to signal contributions that were near or below thesensitivity of the instrument 5. However, the span of highconcentrations for the relatively large ligand, T4L (16.7 kDa), producesa linear response with increased amounts of free T4L-L99A. To confirmthis interpretation, a calibration curve of T4L-L99A was constructed todetect the response of BSI to free concentrations of ligand (FIG. 26c ).The slope of the calibration curve matches (Δm=2.3%) the slope of theascending starting values from αB-D3⋅T4L-L99A binding traces (y @t0—FIG.26c ). This indicates the linear rise of end point values at highconcentrations is due to an increase in free T4L-L99A. Baselinesubtraction accounts for this slope in the steady-state data and wasperformed prior to further data analysis.

Thermodynamic analysis of binding was carried out on thebaseline-corrected data using a two-mode binding model as described bySathish, et. al [Sathish, H. A., Stein, R. A., Yang, G. & McHaourab, H.S. Mechanism of chaperone function in small heat-shock proteins.Fluorescence studies of the conformations of T4 lysozyme bound toalphaB-crystallin. J Biol Chem 278, 44214-21 (2003)]. The detectedsignal was modeled as arising from two components: free T4L and thesHSP●T4L complex. Binding depletes the free ligand pool and increasesthe contribution of the complex to the signal. Using the two-modebinding formalism, steady-state data can be fit to obtain thedissociation constants, the number of binding sites, and the BSI signalarising from each mode. A simpler single-site binding model was alsoperformed and served as a null hypothesis. F ratio comparison of thesingle and two-site binding formalisms led to a rejection of the nullhypothesis at α=0.05 signifying BSI was detecting a bi-modal molecularinteraction. Further confirmation of two-mode binding was obtained froma phenomenological analysis of the real-time binding data. All kinetictraces were fit to both a single and double exponential by nonlinearleast squares regression. A comparison of the square of residuals fromeach fitting routine confirmed the double exponential nature of thedata. The observed rates, as determined from the iterative fittingprocess, are linear functions of L99A concentration. This dependence ispredicted by the law of mass action and suggests at least two separatekinetic events are being monitored by BSI.

Thermodynamic parameters determined by BSI were compared to resultsobtained from ITC experiments (FIG. 26e,f ) and fluorescent bindingmeasurements in TABLE 1. The calculated low affinity dissociationconstant (KD2) is very similar across platforms. However, calorimetricand fluorescence studies of high affinity binding between αB-D3 andT4L-L99A yielded close to a 20-fold increase in the equilibriumdissociation constant (KD1) when compared to BSI analysis. Withoutwishing to be bound by theory, it is likely that the overestimation ofthe affinity by BSI reflects the limited data set in the range of largemolar ratios between αB-D3 and T4L in FIG. 26C. A thorough analysisusing determination of forward and reverse kinetic rates yield moreconsistent parameters as shown below. For the purpose of thiscomparison, however, steady state data was exclusively used becausekinetic data cannot be obtained from traditional ITC.

TABLE 1 T = 37° C. and pH = 7.2 Data ITC Fluorescence BSI moles of αB-D3used 24 × 10⁻⁹ ~50 × 10⁻⁹  1.5 × 10⁻⁹ moles of L99A used 54 × 10⁻⁹ ~50 ×10⁻⁹ 2.93 × 10⁻⁹ n₁ 0.25 0.24 0.24 K_(D1) (μM) 0.015 0.020 0.001 n₂ 1.11.1 1.1 K_(D2) (μM) 2.44 1.00 3.11

The three methods examined provide consistent levels of binding sincethe high affinity binding sites are saturated and low affinity bindingis comparable. However, BSI analysis was carried out with considerablylower amounts of reagent. ITC and fluorescence required roughly 100nanomoles each of sHSP and T4L, while, BSI experiments consumed nearly20 times less. The reduced consumption of analytes is an intrinsicproperty of BSI and can be valuable in molecular interaction studies.Different constructs or mutated sequences often have lower expressionyields, limiting the number of analyses that can be performed. In fact,the level of T4L expression for double mutants is enough to run only twoexperiments via ITC. The same amount of protein is sufficient forapproximately forty experiments with BSI.

b. Specificity of BSI-Detected Binding: Increased α-Crystallin Affinityfor Destabilized Proteins

The coupled equations (FIG. 26a ) predict that the affinity of sHSP totheir substrates reflect the propensity of the latter to occupynon-native states. This prediction was verified for a number of sHSPusing a set of T4L mutants differing in their free energy of unfoldingbut having similar overall structure to the native state [Matthews, B.W. Structural and genetic analysis of the folding and function of T4lysozyme. Faseb J 10, 35-41 (1996)]. In order to establish thespecificity of detection by BSI and highlight the additional kineticdimension provided by this technique, the interaction between αB-D3 andtwo T4L mutants (FIG. 27a ) having different free energies of unfolding.The double mutant, L99A/A130S, is the most destabilized construct of T4Lwith a ΔGunf equal to 3.5 kcal/mole. T4L-D70N is more stable mutant witha free energy of unfolding equal to 6.8 kcal/mol [Matthews, B. W.Structural and genetic analysis of the folding and function of T4lysozyme. Faseb J 10, 35-41 (1996)].

To determine if BSI is sensitive to possible mixing artifacts, mutantsof T4L were mixed in channels with buffer solution and the kinetictraces of multiple concentrations recorded (FIG. 27b ). With linearityof the traces approaching one, real-time data confirms that BSI signalsobserved in the presence of αB-D3 are not an artifact of mixing, but atrue binding signal. FIG. 27c shows the interference pattern observedfor each T4L mutant and the buffer solution while contained within themicrofluidic channel. The zoomed-in region of the fringe patterndemonstrates the sensitivity of BSI to intrinsic refractive index, witha substantial shift seen between the interference patterns of the T4Lmutants and buffer. Simultaneously, the fringe patterns produced for allT4L mutants studied precisely overlap with point-to-point minimamatching. Thus, differences in BSI signals for differentB-crystallin/T4L-mutants must reflect changes in their interactions andnot merely different BSI signals for different mutants.

The association of αB-D3 with multiple concentrations of T4L wasmonitored in real-time by BSI analysis performed at 25° C., pH 8.0 (FIG.28a,b ). As described above, T4L calibration curves were calculated foreach mutant and used for baseline subtraction of the initial startingvalues. Comparison of corrected steady-state data for αB-D3⋅T4Linteractions reveals a qualitative difference between the BSI signals ofeach mutant (FIG. 28c ). An ˜40% reduction in the magnitude is observedfor D70N relative to L99A/A130S at equivalent αB-D3:T4L ratiossuggesting lower levels of binding. Furthermore, saturation is observedat lower molar ratios of αB-D3 to T4L for the L99A/A130S mutant.Interactions, or lack thereof, between αB-D3 and WT-T4L serve as acontrol. Given the larger ΔG_(UNF) of the WT, the equilibrium fractionof unfolded or non-native conformations is negligible (equation 1).Therefore marginal binding is predicted across the entire concentrationrange. Experimentally, BSI signals for αB-D3⋅T4L-WT were within theexperimental noise level.

In order to quantitatively describe the kinetics of the αB-D3⋅T4L systemobserved by BSI, a more robust analysis was developed to overcome theshortcomings of the kinetic analysis performed in earlier BSI studies.Bornhop, et. al [Bornhop, D. J. et al. Free-solution, label-freemolecular interactions studied by back-scattering interferometry.Science 317, 1732-6 (2007)] fit real-time traces to a single exponentialregardless of the complexity of the system. This was mandated byderivation of a first order, ordinary differential equation (ODE) basedon pseudo-first order kinetics under conditions of excess ligandconcentration. Given the two-mode binding of T4L by α-crystallin, such arestricted parametric approach is not adequate for quantitative dataanalysis. Complex systems are more accurately described by higherordered functions, generally a double exponential for interactionstudies. Although extraction of multiple, highly linear rates from asingle kinetic trace is feasible, considering the overall complexity ofthe sHSP molecular interaction system and the number of parameterscorresponding to these interactions a more statistically sound approachin determining equilibrium dissociation constant(s) is needed. Forinstance, in the presence of molar excess of substrate, the contributionof low affinity mode to binding is marginal and the correspondingkinetic trace provides little constraints on the parameters associatedwith that mode.

Therefore, all the traces were fit simultaneously to one set ofparameters that minimizes a global error function, χ2□ Analysis of thekinetic data is based on simple rate equations derived from the bindingequilibria. The time dependent BSI signal was fit to obtain forward(k_(f)) and reverse (k_(r)) rate constants. Commonly referred to asglobal analysis [Beechem, J. M. Expanding time scales usher in a new erafor kinetic studies. Biophys J 74, 2141 (1998)], this strategy reducesthe effects of instrumental artifacts in a particular kinetic trace onthe final parameters and overcomes the uneven sensitivity to theparameters at either extreme of molar ratios.

Results from the global analysis of BSI experiments (TABLE 2) indicatethat the affinity of αB-D3 binding in the high affinity mode increasesby a factor of 4 for the more destabilized mutant, L99A/A130S. Theinteraction of αB-D3 with both constructs of T4L in the low affinitymode appears similar and weak for both mutants. These results clearlyindicate that BSI is capable of measuring molecular interactions withhigh specificity, resolving binding of a sHSP to structurally homologousT4L mutants differing only in their unfolding energies. Furthermore,comparison of the data obtained from the global analysis of BSI kineticdata is consistent with previously published fluorescent analyses of thesame sHSP●T4L binding systems [Koteiche, H. A. & McHaourab, H. S.Mechanism of chaperone function in small heat-shock proteins.Phosphorylation-induced activation of two-mode binding inalphaB-crystallin. J Biol Chem 278, 10361-7 (2003)].

TABLE 2 T4 Lysozyme Data WT D70N L99A-A130S ?G_(unf) (kcal/mol) 14 9.27.9 k_(f1) (M⁻¹s⁻¹) UND 25545 58515 k_(r1) (s⁻¹) UND 1.52 × 10⁻² 5.03 ×10⁻⁶ K_(D1) UND 0.595 μM 0.132 μM k_(f2) (M⁻¹s⁻¹) UND 2342 2970 k_(r1)(s⁻¹) UND 6.82 × 10⁻² 0.115 K_(D2) UND 29.1 μM 33.0 μM ID ? undetectable

c. Interaction of an α-Crystallin Mutant with Another Lens ProteinβB1-Crystallin

The molecular targets of α-crystallin are lens proteins such as β andγ-crystallins. These proteins are long lived and undergo extensivepost-translational modifications; some of which reduce the free energyof unfolding and hence are expected to trigger binding to α-crystallin.A detailed study of the energetics of α- and β-crystallin interactionvia bimane fluorescence of labeled β-crystallin has been reported[McHaourab, H. S., Kumar, M. S. & Koteiche, H. A. Specificity ofalphaA-crystallin binding to destabilized mutants of betaB1-crystallin.FEBS Lett 581, 1939-43 (2007)]. Results from this previous binding studydemonstrate that α-crystallins have low affinity even for highlydestabilized β-crystallins.

Here, BSI was used to evaluate binding of a αA-crystallin mutantgenetically-linked to hereditary cataract to β-crystallin. Mackay, et.al [Mackay, D. S., Andley, U. P. & Shiels, A. Cell death triggered by anovel mutation in the alphaA-crystallin gene underlies autosomaldominant cataract linked to chromosome 21q. Eur J Hum Genet 11, 784-93(2003)] identified a missense mutation in the HspB4 gene on chromosome21q causing the formation of nuclear cataract. The point mutationunderlying cataractogenesis was determined to be a replacement ofarginine for cysteine at residue 49 (αA-R49C). αA-R49C was the firstmutation found outside the conserved α-crystallin domain which gave riseto autosomal dominant cataract. Evidence from cellular studies (Mackey,et al.) suggest the deleterious effect of the mutated protein is causedby a toxic gain of function. αA-R49C was reported to have substantiallyhigher affinity to destabilized mutants of T4L with Koteiche andMchaourab suggesting that this mutant acts to unfold cellular proteinsforming insoluble substrate-saturated complexes Koteiche, H. A. &McHaourab, H. S. Mechanism of a hereditary cataract phenotype. Mutationsin alphaA-crystallin activate substrate binding. J Biol Chem 281,14273-9 (2006)]. Furthermore, interaction of αA-R49C with thiolatedsubstrates leads to formation of disulfide-linked α-crystallin dimers[Kumar, M. S., Koteiche, H. A., Claxton, D. P. & McHaourab, H. S.Disulfide cross-links in the interaction of a cataract-linkedαA-crystallin mutant with bB1-crystallin. FEBS Lett Submitted (2008)].

To highlight the utility of BSI in monitoring native protein-proteininteractions, binding studies of the cataract-linked mutant, αA-R49C,were performed with the lens protein βB1-crystallin at physiological pHand at 37° C. To mimic thiolation in the lens, βB1-crystallin waslabeled with a monobromobimane probe via a disulfide linkage. Otherexperimental conditions and the optical setup were kept consistent withthe sHSP studies described above. A similar system was studied by Kamei,et. al [Kamei, A. & Matsuura, N. Analysis of crystallin-crystallininteractions by surface plasmon resonance. Biol Pharm Bull 25, 611-5(2002)] with the use of SPR. However, as is compulsory for SPR studies,αA-crystallin was immobilized onto a gold substrate. β-crystallin waspassed over the substrate bound αA-crystallin for 25 minutes at a flowrate of 2 μL/min with changes in the local refractive index near thegold substrate used to monitor binding. In addition, regeneration of theSPR surface was accomplished by introduction of a 15 μL solution of 0.1Msodium acetate and 0.15M sodium chloride at an extreme pH of 2.3.

Multiple concentrations of βB1-crystallin were assayed against aconstant concentration of the mutated αA-crystallin (FIG. 29a ). Globalanalysis was used to analyze the αA-R49C●βB1-crystallin interactionmonitored by BSI. Kinetic data determined for one binding mode wasquantifiable by both BSI and SPR, giving similar values for k_(f) andk_(r), varying by 20% and 33% respectively (FIG. 29c ). However, SPR wasunable to distinguish a high affinity binding mode evident in theanalysis of the BSI data. The inability of SPR to detect a second ratecould be due to α-crystallin being immobilized on the gold surface. BSIresults for the αA-R49C●βB1-crystallin were found to be in agreementwith steady-state analysis of BSI data (FIG. 29b ) as well asfluorescent data published previously by Koteiche, et. al [Shi, J.,Koteiche, H. A., McHaourab, H. S. & Stewart, P. L. Cryoelectronmicroscopy and EPR analysis of engineered symmetric and polydisperseHsp16.5 assemblies reveals determinants of polydispersity and substratebinding. J Biol Chem 281, 40420-8 (2006)].

d. General Methods

Site-directed mutagenesis of T4L32, αA-crystallin [Berengian, A. R.,Parfenova, M. & McHaourab, H. S. Site-directed spin labeling study ofsubunit interactions in the alpha-crystallin domain of small heat-shockproteins. Comparison of the oligomer symmetry in alphaA-crystallin, HSP27, and HSP 16.3. J Biol Chem 274, 6305-14 (1999)], αB-crystallin[Koteiche, H. A. & McHaourab, H. S. Mechanism of chaperone function insmall heat-shock proteins. Phosphorylation-induced activation oftwo-mode binding in alphaB-crystallin. J Biol Chem 278, 10361-7 (2003),and β-crystallin [Sathish, H. A., Koteiche, H. A. & McHaourab, H. S.Binding of destabilized betaB2-crystallin mutants to alpha-crystallin:the role of a folding intermediate. J Biol Chem 279, 16425-32 (2004)]has been described previously. Protein expression was carried out incompetent BL21 (sHSP) or K38 (T4L) cell lines. Cells were transformedwith mutant plasmids and cultured in Luria-Burtani (LB) broth containingsmall concentrations of ampicillin overnight at 32° C. This seed culturewas then increased in volume and incubated for 2-3 hours at 37° C. untilmid-log phase was reached. After cooling to room temperature, proteinexpression was induced by the addition of 0.4 mM isopropylβ-D-thioglactopyranoside (IPTG). Protein expression was carried out for3 hours at 32° C. and 2 hours at ˜30° C. post-induction for sHSP and T4Lrespectively.

All T4L mutants were purified by a two step separation process. Cationexchange using a Resource S column was used as an initial clean-up step.Immediately following elution, T4L mutants were labeled withmonobromobimane in a 10 fold stoichiometric ratio. Reaction of thefluorophore with cysteine at position 151 was allowed to proceedovernight to ensure complete derivatization. Although neither BSI norcalorimetry require a labeled analyte for detection, all T4L mutantswere fluorescently derivatized and used throughout the experiments toensure no variability existed between the ligands used in each detectionmethod. Labeled solutions were further purified by size exclusionchromatography using a Superdex 75 column. The eluted analyte wasconcentrated using Amicon® centrifugal concentrators and characterizedby UV-Vis spectroscopy. Labeling efficiency was determined by monitoringabsorbance peaks at 280 and 380 nm. Mutant T4L concentrations weredetermined by absorbance at 280 nm using an extinction coefficient of1.231 cm²/mg.

αB-crystallin was purified in a three step separation process.αB-crystallin was loaded onto a Source Q column for anion exchange andeluted with a sodium chloride gradient. After the eluent had beenadjusted to a final concentration of 0.5M ammonium sulfate, the solutionwas loaded onto a phenyl-Sepharose column and eluted with a gradienttransitioning from 1M to 0M ammonium sulfate. A final purification stepwas performed by size-exclusion chromatography using a Superose 6column. No phenyl-Sepharose column was used in the purification ofβ-crystallin or αA-crystallin. Following anion exchange, purifiedβ-crystallin was reacted with a 10-fold molar excess of bimane label andincubated for 2 hours at room temperature. Excess bimane label wasremoved and the fluorescently labeled β-crystallin was purified bysize-exclusion chromatography on a Superdex 75 column. Solutions of sHSPwere concentrated using centrifugal filters and then characterized byUV-Vis spectroscopy. The concentration of each sHSP construct wasdetermined at 280 nm using the appropriate extinction coefficient.

BSI. Solutions of T4L and sHSP variants used in BSI experiments werebuffered with 9 mM Tris, 6 mM MES, 500 mM NaCl, and 0.2% sodium azide.The pH of each solution was adjusted by the addition of small amounts of5N NaOH or 5N HCl and monitored by a standard pH electrode. Allsolutions were filtered and degassed prior to binding experiments.Solutions were kept on ice during the experiment and briefly allowed towarm to room temperature prior to their introduction into themicrochannel. The mixing chip design used was a hybridserpentine-hydrodynamic focusing mixer as described by Bornhop, et. al[Bornhop, D. J. et al. Free-solution, label-free molecular interactionsstudied by back-scattering interferometry. Science 317, 1732-6 (2007)].Experiments were maintained at the desired temperature by a MELCORtemperature controller coupled to a Peltier device. Approximately 4 μLof each sHSP and T4L construct was used in obtaining a singleassociation curve. Binding experiments were monitored in real-time atfrequencies approximately 50-100 Hz and in detection volumes on theorder of picoliters.

A MicroCal VP-ITC (Isothermal Titration calorimetry) was employed tocross validate results obtained by BSI. Solutions of αB-crystallin andT4L-L99A were buffered with 0.15M Na2H2PO4, 0.1M KCl, 0.1 mM EGTA, and0.1% sodium azide. Solutions were kept on ice before the experiment andallowed to warm near the experimental temperature prior to sampleintroduction. All solutions were filtered through a 0.2 micron disc anddegassed before calorimetric experiments. αB-crystallin (˜1.4 mL) washoused in the sample cell for ITC experiments and had an initialconcentration of 12 μM. The buffer solution was kept in the referencecell. Approximately 260 μL of T4L mutant at a concentration equal to 120μM was drawn into a syringe housed within an automated pipette system.The syringe was placed in the sample cell and spun at 300 RPM. Thesystem was allowed to equilibrate for roughly 2 hours. Once no drift wasobserved in the baseline and the temperature remained fairly constant,an automated injection sequence was initiated. 104 of the T4L mutant wasinjected into the sample cell containing αB-crystallin twenty-five timeswith ˜7 minutes allowed between injections to bring the signal back tobaseline. The heat evolved after each injection was recorded andexperimental data was analyzed by Origin software to calculatethermodynamic parameters for comparison to BSI and fluorescenceexperiments.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otheraspects of the invention will be apparent to those skilled in the artfrom consideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

What is claimed is:
 1. A method comprising detecting molecularinteraction between analytes in free-solution wherein at least one ofthe analytes is label-free and detection is performed by back-scatteringinterferometry.
 2. The method of claim 1, wherein at least two analytesare label-free.
 3. The method of claim 1, wherein the interaction is abiomolecular interaction.
 4. The method of claim 1, wherein the analytesare present in a sample in a microfluidic channel in a substrate andback-scattering interferometry comprises directing a coherent light beamonto the substrate such that the light beam is incident on the channelto generate backscattered light through reflective and refractiveinteraction of the light beam with a substrate channel interface and thesample, the backscattered light comprising interference fringe patternsincluding a plurality of spaced light bands whose positions shift inresponse to changes in the refractive index of the fluid sample.
 5. Themethod of claim 1, wherein the interaction is a binding event betweenone or more of antibody-antigen, protein-protein, small molecule-smallmolecule; small molecule-protein, drug-receptor; antibody-cell;protein-cell; oligonucleotide-cell; carbohydrate-cell; cell-cell;enzyme-substrate; protein-DNA; protein-aptamer; DNA-DNA; RNA-RNA;DNA-RNA; protein-RNA; small molecule-nucleic acid; biomolecule-molecularimprint; biomolecule-protein mimetic; biomolecule-antibody derivatives;lectin-carbohydrate; biomolecule-carbohydrate; small molecule-micelle;small molecule-cell membrane; and enzyme-substrate.
 6. The method ofclaim 1, wherein the substrate and channel together comprise a capillarytube.
 7. The method of claim 1, wherein the substrate and channeltogether comprise a microfluidic device.
 8. A method for real-time,free-solution determination of molecular interactions comprising thestep of detecting the formation of one or more interaction products oftwo unlabeled, non-immobilized analytes, wherein at least one of theanalytes is present during the determination at a concentration of lessthan about 5.0×10⁻⁵M and/or wherein at least one of the analytes ispresent during the determination in a solution with a volume in thedetection zone of less than about 500 nL.
 9. The method of claim 8,wherein at least two analytes are label-free.
 10. The method of claim 8,wherein one of the analytes is present in a concentration of less thanabout 5.0×10⁻⁷M.
 11. The method of claim 8, wherein one of the analytesis present in a concentration of less than about 5.0×10⁻⁹M.
 12. Themethod of claim 8, wherein the interaction is a biomolecularinteraction.
 13. The method of claim 8, wherein the volume in thedetection zone of less than about 300 nL.
 14. The method of claim 8,wherein the volume in the detection zone of less than about 100 nL. 15.A detection system comprising: (a) a microfluidic channel formed in asubstrate; (b) a solution comprising label-free analytes in freesolution in the channel; and (c) an interferometer that detectsmolecular interactions between the analytes in the channel.
 16. Thesystem of claim 15, wherein the detector is an interferometer.
 17. Thesystem of claim 15, wherein the interaction is a biomolecularinteraction.
 18. The system of claim 15, wherein one of the analytes ispresent in a concentration of less than about 5.0×10⁻⁵M.
 19. The systemof claim 15, wherein the volume in the detection zone of less than about500 nL.
 20. The system of claim 15, wherein one of the analytes ispresent in a concentration of less than about 5.0×10⁻⁵M and wherein thevolume in the detection zone of less than about 500 nL.